BR112016003339B1 - ENZYME COMPOSITION, AND, PROCESS OF PRODUCING A FERMENTATION PRODUCT FROM STARCH CONTAINING MATERIAL - Google Patents

Abstract

ENZYME COMPOSITION, AND, PROCESS OF PRODUCING A FERMENTATION PRODUCT FROM STARCH CONTAINING MATERIAL. The invention relates to enzyme compositions comprising a glucoamylase, an alpha-amylase, and optionally a cellulolytic composition and/or a protease. The invention also relates to the use thereof in processes for producing sugars and/or fermentation products from starch-containing material by saccharifying and/or fermenting the starch-containing material at a temperature below the initial gelatinization temperature.

Description

TECHNICAL FIELD

[001] The present invention is directed to enzymatic compositions and processes for producing sugars and/or fermentation products from starch-containing materials using such enzymatic compositions.

PRIOR TECHNIQUE

[002] Processes for producing sugars and fermentation products, such as ethanol, from starch-containing materials are well known in the art and are currently used commercially.

[003] When producing fermentation products such as ethanol, starch is conventionally converted to dextrins using a liquefactor enzyme (for example, Bacillus alpha-amylase), at temperatures above the initial gelatinization temperature of starch above 100 °C. The generated dextrins are hydrolyzed to sugars using a saccharification enzyme (eg glucoamylase) and fermented to the desired fermentation product using a fermenting organism such as a yeast strain derived from Saccharomyces cerevisiae. Typically hydrolysis and fermentation are done in a simultaneous saccharification and fermentation (SSF) step.

[004] Another type of process is also used commercially today. Starch is converted to dextrins at temperatures below the initial gelatinization temperature of starch and hydrolyzed to sugars during SSF. This type of process is referred to as a crude starch hydrolysis (RSH) process, or alternatively a "one-step process" or a "no-bake" process.

[005] Document US4514496 relates to a process for producing alcohol by mixing a ground starch material with mash liquid without cooking, adding a saccharification enzyme preparation derived from a Rhizopus sp., and adding a alcoholic fermenting yeast, and the fermentation of the paste.

[006] Document WO2003/066826 discloses a method for producing an alcohol by contacting a carbon substrate and at least one substrate conversion enzyme to produce an intermediate, and contacting said intermediate with at least one enzyme intermediate converter.

[007] WO 2004/080923 relates to a process for producing an alcohol product comprising the sequential steps of providing a paste comprising water and granular starch; stopping said paste in the presence of an acid alpha-amylase and a glucoamylase, at a temperature of 0°C to 20°C below the initial gelatinization temperature of said granular starch for a period of 5 minutes to 12 hours; stopping said paste in the presence of an acid alpha-amylase and a glucoamylase and a yeast at a temperature between 10°C and 35°C for a period of 20 to 250 hours, in order to produce ethanol. A xylanase, a cellulase and a phytase may be present during the stopping steps.

[008] Document WO2004/081193 relates to a process for producing ethanol from plant material, comprising reducing the plant material to produce material comprising starch, saccharifying the starch, without cooking, with an enzyme composition, and fermenting the incubated starch to obtain a composition comprising at least 15 vol -% ethanol.

[009] Document WO2006/069289 relates to glucoamylases derived from Trametes cingulata, Pachykytospora papyracea, and Leucopaxillus giganteus and their use in a process for producing a fermentation product from starch-containing material at a temperature lower than the initial gelatinization temperature.

[0010] WO 2011/068803 discloses glucoamylase derived from Gloeophyllum sepiarium, Gloeophyllum trabeum, or Gloeophyllum abietinum and its use in processes for producing a fermentation product from starch-containing material.

[0011] The present invention relates to enzymatic compositions suitable for crude starch hydrolysis processes and the use of these enzymatic compositions in these processes.

SUMMARY OF THE INVENTION Enzymatic composition of the invention

[0012] The invention relates to the enzyme composition comprising a number of enzymatic activities and to its use in processes for the production of sugars and/or fermentation products, such as, in particular, ethanol. The enzyme composition of the invention can be a mixture of a number of different enzyme activities. The enzymatic activities can have the same origin or different origins.

[0013] In the first aspect, the present invention relates to compositions comprising glucoamylase and alpha-amylase, and optionally protease.

[0014] In a preferred embodiment, the enzyme composition comprises Gloeophyllum glucoamylase, preferably Gloeophyllum trabeum glucoamylase and an alpha-amylase.

[0015] In a preferred embodiment, the alpha-amylase is derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably the one disclosed as V039 in Table 5 in WO 2006/069290, or SEQ ID NO: 13 shown in this document.

[0016] In an especially preferred embodiment, the enzyme composition comprises Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18, shown herein having one or more of the following substitutions: S95P, A121P, especially S95P+A121P; and an alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably as shown in SEQ ID NO: 13, disclosed herein, preferably having one or more of the following substitutions : G128D, D143N, especially G128D+D143N.

[0017] In another embodiment the enzyme composition comprises a glucoamylase derived from a strain of the genus Pycnoporus, in particular a strain of Pycnoporus sanguineus, such as a strain described in WO 2011/066576 (SEQ ID NOs 2, 4 or 6) . In a preferred embodiment, the enzyme composition comprises the glucoamylase shown in SEQ ID NO: 4 in WO 2011/066576 or in SEQ ID NO: 17 shown herein and an alpha-amylase.

[0018] In a preferred embodiment, the alpha-amylase is derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably the one disclosed as V039 in Table 5 in WO 2006/069290, or SEQ ID NO: 13 shown in this document.

[0019] In an especially preferred embodiment, the enzyme composition comprises the Pycnoporus sanguineus glucoamylase shown in SEQ ID NO: 17 herein, or a glucoamylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17 shown herein, and an alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and binding domain starch (SBD), preferably that shown in SEQ ID NO: 13 set forth herein, or an alpha-amylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13 set forth herein preferably having one or more of the following substitutions: G128D, D143N, especially G128D+D143N.

[0020] In a preferred embodiment, the present invention relates to compositions comprising i) glucoamylase; ii) alpha-amylase; iii) cellulolytic enzyme composition; iv) optionally protease.

[0021] In an especially preferred embodiment, the enzyme composition comprises Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 herein, or a glucoamylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18, preferably having one or more of the following substitutions: S95P, A121P, especially S95P+A121P; and an alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably that shown in SEQ ID NO: 13 set forth herein, or an alpha-amylase having at least 60% , at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13 shown herein preferably having one or more of the following substitutions: G128D, D143N, especially G128D+D143N, and a cellulolytic enzyme composition derived from Trichoderma reesei further comprising Thermoascus aurantiacus GH61 polypeptide having enhancing cellulolytic activity (e.g. SEQ ID NO: 2 in WO 2005/074656 or in SEQ ID NO: 9 shown herein) and beta-glucosidase from Aspergillus fumigatus (e.g. SEQ ID NO: 2 of WO 2005/047499 or in SEQ ID NO: 8 set forth herein), or a cellulolytic enzyme composition derived from Trichoderma reesei further comprising GH61A polypeptide from Penicillium emersonii, e.g. that disclosed as SEQ ID NO: 2 in WO 2011/041397 or in SEQ ID NO: 10 set forth herein, and beta-glucosidase from Aspergillus fumigatus, e.g. as SEQ ID NO: 2 in WO 2005/047499 or in SEQ ID NO: 8 set forth herein, or a variant thereof, preferably a variant having one, preferably all, of the following substitutions: F100D, S283G, N456E, F512Y, CBH I from Aspergillus fumigatus, e.g. as disclosed as SEQ ID NO: 6 in WO 2011/057140 and SEQ ID NO: 6 set forth herein, and CBH II from Aspergillus fumigatus, e.g. as disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 shown herein.

[0022] In another especially preferred embodiment, the enzyme composition comprises the Pycnoporus sanguineus glucoamylase shown in SEQ ID NO: 17 herein, or a glucoamylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17, and an alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD ), preferably that shown in SEQ ID NO: 13 set forth herein, or an alpha-amylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85% , at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 % sequence identity with SEQ ID NO: 13, shown herein preferably having one or more of the following substitutions: G128D, D143N, especially G128D+D143N, and a cellulatic composition derived from Trichoderma reesei further comprising Thermoascus aurantiacus GH61 polypeptide having enhancing cellulolytic activity (e.g. SEQ ID NO: 2 in WO 2005/074656 or in SEQ ID NO: 9 set forth herein) and beta-glucosidase from Aspergillus fumigatus (e.g. SEQ ID NO: 2 of WO 2005/ 047499 or SEQ ID NO: 8 set forth herein), or a cellulatic composition derived from Trichoderma reesei preferably further comprising GH61A polypeptide from Penicillium emersonii, for example that disclosed as SEQ ID NO: 2 in WO 2011/041397 or in SEQ ID NO: 10 shown in this document, and beta-glucosidase from Aspergillus fumigatus, e.g. that disclosed as SEQ ID NO: 2 in WO 2005/047499 or in SEQ ID NO: 8 shown in this document, or a variant thereof, preferably a variant having one, preferably all, of the following substitutions: F100D, S283G, N456E, F512Y, CBH I from Aspergillus fumigatus, e.g. as disclosed as SEQ ID NO: 6 in WO 2011/057140 and SEQ ID NO: 6 set forth herein document, and CBH II from Aspergillus fumigatus, e.g. that disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 presented herein.

[0023] In one embodiment a protease is contained in the enzyme composition of the invention. In a preferred embodiment, the protease is a metalloprotease or a serine protease. In one embodiment the enzyme composition comprises a metalloprotease preferably derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670, such as the metalloprotease disclosed as the mature part of SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature polypeptide of SEQ ID NO: 3 disclosed herein.

[0024] In one embodiment, the protease is derived from a strain of Pyrococcus, such as a strain of Pyrococcus furiosus, such as the protease disclosed in SEQ ID NO: 1 in US 6,358,726 or in SEQ ID NO: 5 set forth herein.

[0025] In one embodiment, the protease is the mature sequence of Meripilus giganteus protease 3 (protease of the S53 family of peptidases) discussed in Example 2 in WO 2014/037438 and shown as SEQ ID NO: 20 shown herein. In one embodiment, the protease is the mature sequence of Meripilus giganteus protease 3 shown as SEQ ID NO: 19 herein and SEQ ID No: 5 in WO 2014/037438.

[0026] In a preferred embodiment, the ratio of glucoamylase to alpha-amylase is between 99:1 and 1:2, such as between 98:2 and 1:1, such as between 97:3 and 2:1, such as between 96:4 and 3:1, such as 97:3, 96:4, 95:5, 94:6, 93:7, 90:10, 85:15 (mg PE (Enzyme Protein) glucoamylase: mg of PE (Enzyme Protein) alpha amylase).

Invention Process

[0027] In a second aspect, the invention relates to processes for producing fermentation products, such as especially ethanol, from a starch-containing material, such as granular starch, comprising: (i) saccharifying a material containing starch at a temperature below the initial gelatinization temperature; and (ii) fermentation using a fermentation organism; wherein the saccharification and/or fermentation is carried out in the presence of the following enzymes: glucoamylase and alpha-amylase, and optionally protease.

[0028] In a preferred embodiment, the following enzymes are present and/or are added during saccharification and/or fermentation: the glucoamylase from Gloeophyllum trabeum shown in SEQ ID NO: 18 presented herein, or a glucoamylase having at least 60% , at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 18, preferably one having one or more of the following substitutions: S95P, A121P, especially S95P+A121P, is an alpha-amylase.

[0029] In a preferred embodiment, the alpha-amylase is derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), disclosed as V039 in Table 5 in WO 2006/069290, or in SEQ ID NO: 13 shown in this document.

[0030] In another preferred embodiment, the following enzymes are present and/or are added during saccharification and/or fermentation: the glucoamylase from Gloeophyllum trabeum shown in SEQ ID NO: 18 presented herein, or a glucoamylase having at least 60% , at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 18 set forth herein, preferably one having one or more of the following substitutions: S95P, A121P, especially S95P+A121P, and alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably that shown in SEQ ID NO: 13 shown herein, or an alpha-amylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13 shown in this document, preferably one having one or more of the following substitutions: G128D, D143N, especially G128D+143N.

[0031] In another preferred embodiment, the following enzymes are present and/or are added during saccharification and/or fermentation: the Pycnoporus sanguineus glucoamylase shown in SEQ ID NO: 17 presented herein and an alpha-amylase.

[0032] In a preferred embodiment, the alpha-amylase is derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably the one disclosed as V039 in Table 5 in WO 2006/069290, or SEQ ID NO: 13 shown herein, preferably one having one or more of the following substitutions: G128D, D143N, especially G128D+D143N.

[0033] In one embodiment, a protease is present and/or is added during saccharification and/or fermentation. In a preferred embodiment, the protease is a metalloprotease or a serine protease. In one embodiment the metalloprotease is preferably derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670, such as the metalloprotease disclosed as the mature part of SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature polypeptide of SEQ ID NO: 3 disclosed herein.

[0034] In a preferred embodiment, the protease is a protease from the S53 family of peptidases derived from a strain of Meripilus, preferably a strain of Meripilus giganteus. In one embodiment, the protease is the mature sequence of Meripilus giganteus protease 3 (protease of the S53 family of peptidases) discussed in Example 2 in WO 2014/037438 and shown as SEQ ID NO: 20 herein. In one embodiment, the protease is the mature sequence of Meripilus giganteus protease 3 shown as SEQ ID NO: 19 herein and SEQ ID No: 5 in WO 2014/037438. In a preferred embodiment, the invention relates to processes for producing fermentation products, such as especially ethanol, from a starch-containing material, such as granular starch, comprising: (i) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and (ii) fermentation using a fermentation organism; wherein the saccharification and/or fermentation is carried out in the presence of the following enzymes: i) glucoamylase; ii) alpha-amylase; iii) cellulotic enzyme composition; iv) optionally protease.

[0035] In another preferred embodiment, the following enzymes are present and/or are added during saccharification and/or fermentation: the glucoamylase from Gloeophyllum trabeum shown in SEQ ID NO: 18, presented in this document, preferably the one having one or more of the following substitutions: S95P, A121P, especially S95P+A121P, and alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably that shown in SEQ ID NO: 13, shown herein document, preferably one having one or more of the following substitutions: G128D, D143N, especially G128D+143N, and a cellulolytic enzyme composition derived from Trichoderma reesei, preferably further comprising Thermoascus aurantiacus GH61 polypeptide having enhancing cellulolytic activity (e.g., SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 9 set forth herein) and Aspergillus fumigatus beta-glucosidase (e.g. SEQ ID NO: 2 of WO 2005/047499 or SEQ ID NO: 8 set forth herein), or a cellulolytic enzyme composition derived from Trichoderma reesei further comprising GH61A polypeptide from Penicillium emersonii, for example that disclosed as SEQ ID NO: 2 in WO 2011/041397 or in SEQ ID NO: 10 set forth herein, and beta-glucosidase from Aspergillus fumigatus, e.g. that disclosed as SEQ ID NO: 2 in WO 2005/047499 or in SEQ ID NO: 8 set forth herein, or a variant thereof, preferably a variant having one, preferably all, of the following substitutions: F100D, S283G, N456E, F512Y, CBH I from Aspergillus fumigatus, e.g. the one disclosed as SEQ ID NO: 6 in WO 2011/057140 and SEQ ID NO: 6 set forth herein, and CBH II from Aspergillus fumigatus , e.g. that disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 presented herein.

[0036] In one embodiment, a protease is present and/or is added during saccharification and/or fermentation. In a preferred embodiment, the protease is a metalloprotease or a serine protease. In one embodiment the metalloprotease is preferably derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670, such as the metalloprotease disclosed as the mature part of SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature polypeptide of SEQ ID NO: 3 disclosed herein.

[0037] In a preferred embodiment, the protease is a protease from the S53 family of peptidases derived from a strain of Meripilus, preferably a strain of Meripilus giganteus. In one embodiment, the protease is the mature sequence of Meripilus giganteus protease 3 (protease of the S53 family of peptidases) discussed in Example 2 in WO 2014/037438 and shown as SEQ ID NO: 20 set forth herein, or a protease having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20. In a preferred embodiment, the protease is the mature sequence of protease 3 from Meripilus giganteus shown as SEQ ID NO: 19 in this document and SEQ ID No: 5 in WO 2014/037438.

[0038] In a preferred embodiment, the ratio of glucoamylase to alpha-amylase is between 99:1 and 1:2, such as between 98:2 and 1:1, such as between 97:3 and 2:1, such as between 96:4 and 3:1, such as 97:3, 96:4, 95:5, 94:6, 93:7, 90:10, 85:15, 83:17 or 65:35 (mg of PE glucoamylase: mg of PE alpha amylase).

[0039] In a preferred embodiment, the total dose of glucoamylase and alpha-amylase is 10-1,000 µg/g of DS, such as 50-500 µg/g of DS, such as 75-250 µg/g of DS.

[0040] In a preferred embodiment, the total dose of added cellulolytic enzyme composition is 10-500 µg/g DS, such as 20400 µg/g DS, such as 20-300 µg/g DS.

[0041] In one embodiment, the dose of protease added is 1-200 µg/g DS, such as 2-100 µg/g DS, such as 3-50 µg/g DS.

[0042] In a preferred embodiment, the saccharization step (a) and the fermentation step (b) are carried out simultaneously. In a preferred embodiment an enzyme composition described above is used in a process of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The invention relates to the enzyme composition comprising a number of enzymatic activities and to its use in processes for the production of sugars and/or fermentation products, such as, in particular, ethanol. An enzyme composition of the invention is suitable for use in processes for producing sugars and/or fermentation products carried out as a raw starch hydrolysis process (i.e. no cooking process) at temperatures below the initial gelatinization temperature of the starch in question. When using an enzyme composition of the invention in a crude starch hydrolysis process the yield of the fermentation product such as ethanol is improved when compared to a corresponding process where the known enzyme composition consisting of glucoamylase is used and alpha-amylase. In a preferred embodiment, the yield of the fermentation product, such as ethanol, is improved when compared to a corresponding process where glucoamylase or alpha-amylase alone is used or where an enzyme composition consisting of glucoamylase from Trametes cingulata is used and alpha-amylase from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD).

[0044] In the first aspect, the present invention relates to compositions comprising glucoamylase and alpha-amylase, and optionally protease.

[0045] In a preferred embodiment, the enzyme composition comprises a Gloeophyllum glucoamylase, preferably Gloeophyllum trabeum glucoamylase, especially the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18, and an alpha-amylase.

[0046] In one embodiment, the glucamylase is derived from Gloeophyllum trabeum, such as that shown in SEQ ID NO: 18 set forth herein, or a glucoamylase selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 18 shown in this document; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 18 presented in this document.

[0047] In a preferred embodiment, the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18, presented herein has one of the following substitutions: V59A; S95P; A121P; T119W; S95P+A121P; V59A+S95P; S95P+T119W; V59A+S95P+A121P; or S95P+T119W+A121P, especially S95P+A121P.

[0048] In a preferred embodiment, the alpha-amylase is derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably the one disclosed as V039 in Table 5 in WO 2006/069290 , or in SEQ ID NO: 13 shown herein, or an alpha-amylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity sequence with SEQ ID NO: 13 shown in this document.

[0049] In one embodiment the alpha-amylase is Rhizomucor pusillus alpha-amylase or Rhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), especially one having at least least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S + Y141W; A76G + Y141W; G128D + Y141W; G128D + D143N; P219C + Y141W; N142D + D143N; Y141W + K192R; Y141W + D143N; Y141W + N383R; Y141W + P219C + A265C; Y141W + N142D + D143N; Y141W + K192R V410A; G128D + Y141W + D143N; Y141W + D143N + P219C; Y141W + D143N + K192R; G128D + D143N + K192R; Y141W + D143N + K192R + P219C; G128D + Y141W + D143N + K192R; or G128D + Y141W + D143N + K192R + P219C, especially G128D+D143N (using SEQ ID NO: 13 shown in this document for numbering).

[0050] In one embodiment the enzyme composition comprises glucoamylase from Gloeophyllum trabeum, preferably that shown in SEQ ID NO: 18, and an alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain ( SBD), disclosed as V039 in Table 5 in WO 2006/069290, or in SEQ ID NO: 13 presented herein.

[0051] In another preferred embodiment, the enzyme composition comprises glucoamylase from Gloeophyllum trabeum, preferably that shown in SEQ ID NO: 18, shown herein, especially one having one or more of the following substitutions: S95P, A121P, especially S95P+A121P (using SEQ ID NO: 13 shown in this document for numbering); and alpha-amylase derived from Rhizomucor pusillus preferably with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably as shown in SEQ ID NO: 13 set forth herein, preferably one having one or more of the following replacements: G128D, D143N, especially G128D+D143N (using SEQ ID NO: 13 shown in this document for numbering).

[0052] In another embodiment the enzyme composition comprises a glucoamylase derived from a strain of the genus Pycnoporus, in particular a strain of Pycnoporus sanguineus, such as a strain described in WO 2011/066576 (SEQ ID NOs 2, 4 or 6) . In a preferred embodiment, the enzyme composition comprises the glucoamylase shown in SEQ ID NO: 4 in WO 2011/066576 or in SEQ ID NO: 17 shown herein, or a glucoamylase having at least 60%, at least 70%, p .eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17 shown herein, and an alpha-amylase.

[0053] In a preferred embodiment, the alpha-amylase is derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably that disclosed as V039 in Table 5 in WO 2006/069290 , or in SEQ ID NO: 13 shown herein, or an alpha-amylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity sequence with SEQ ID NO: 13 shown in this document.

[0054] In an especially preferred embodiment, the enzyme composition comprises the Pycnoporus sanguineus glucoamylase shown in SEQ ID NO: 17 herein; or a glucoamylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17 shown herein, and alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably that shown in SEQ ID NO: 13 shown herein, or an alpha-amylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13 shown herein preferably having one or more of the following substitutions : G128D, D143N, especially G128D+D143N.

[0055] In a preferred embodiment, the present invention relates to compositions comprising i) glucoamylase; ii) alpha-amylase; iii) cellulotic enzyme composition; optionally iv) protease.

[0056] In an especially preferred embodiment, the enzyme composition comprises the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 herein having preferably one or more of the following substitutions: S95P, A121P, especially S95P+A121P; and alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably as shown in SEQ ID NO: 13, disclosed herein, preferably having one or more of the following substitutions : G128D, D143N, especially G128D+D143N, and a cellulolytic enzyme composition derived from Trichoderma reesei, preferably further comprising Thermoascus aurantiacus GH61 polypeptide having enhancing cellulolytic activity (SEQ ID NO: 2 in WO 2005/074656 or in SEQ ID NO: 9 disclosed herein) and beta-glucosidase from Aspergillus fumigatus (SEQ ID NO: 2 of WO 2005/047499 or in SEQ ID NO: 8 disclosed herein), or a cellulolytic enzyme composition derived from Trichoderma reesei further comprising GH61A polypeptide from Penicillium emersonii, disclosed as SEQ ID NO: 2 in WO 2011/041397 or in SEQ ID NO: 10 set forth herein, and beta-glucosidase from Aspergillus fumigatus disclosed as SEQ ID NO: 2 in WO 2005/047499 or in SEQ ID NO : 8 disclosed herein, or a variant thereof, preferably a variant having one, preferably all, of the following substitutions: F100D, S283G, N456E, F512Y, Cel7A CBHI from Aspergillus fumigatus disclosed as SEQ ID NO: 6 in WO 2011/057140 and SEQ ID NO: 6 shown herein and CBH II from Aspergillus fumigatus disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 shown herein.

[0057] In one embodiment the enzyme composition comprises a metalloprotease preferably derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670, such as the metalloprotease disclosed as the mature part of SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature polypeptide of SEQ ID NO: 3 disclosed herein, or a protease having at least 60%, at least 70% , eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3 shown herein.

[0058] In a preferred embodiment, the protease is a protease from the S53 family of peptidases derived from a strain of Meripilus, preferably a strain of Meripilus giganteus. In one embodiment, the protease is the mature sequence of Meripilus giganteus protease 3 (protease of the S53 family of peptidases) discussed in Example 2 in WO 2014/037438 and shown as SEQ ID NO: 20 set forth herein, or a protease having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 20 shown herein. In one embodiment, the protease is the mature sequence of Meripilus giganteus protease 3 shown as SEQ ID NO: 19 in this document and SEQ ID No: 5 in WO 2014/037438, or a protease having at least 60%, at least 70% , eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 19 shown herein.

[0059] Other enzymes may also be present. Specifically contemplated enzyme components are described further below.

Cellulolytic Enzyme Composition

[0060] An enzyme composition of the invention optionally contains a cellulolytic enzyme composition. The cellulolytic enzyme composition consists of or comprises one or more cellulolytic enzymes. The cellulolytic enzyme composition can be from any source. In a preferred embodiment, the cellulolytic enzyme composition comprises cellulolytic enzyme of fungal origin.

[0061] In one embodiment, the cellulolytic enzyme composition is derived from a strain of Trichoderma, such as Trichoderma reesei; or a Humicola strain, such as Humicola insolens; or a strain of Chrysosporium, such as Chrysosporium lucknowense; or a strain of Penicillium, such as Penicillium decumbens. In a preferred embodiment, the cellulolytic enzyme composition is derived from a strain of Trichoderma reesei.

[0062] The cellulolytic enzyme composition may comprise a beta-glucosidase, a cellobiohydrolase, and an endoglucanase.

[0063] In one embodiment the cellulolytic enzyme composition comprises one or more polypeptides selected from the group consisting of: - beta-glucosidase; - cellobiohydrolase I; - cellobiohydrolase II; or a mixture thereof.

[0064] In a preferred embodiment, the cellulolytic enzyme composition further comprises a GH61 polypeptide having enhancing cellulolytic activity. Enhanced cellulolytic activity. is defined and determined as described in WO 2011/041397 (incorporated herein by reference).

[0065] The term "GH61 polypeptide having enhancing cellulolytic activity" means a GH61 polypeptide which enhances the hydrolysis of a cellulosic material by enzymes having cellulolytic activity. For purposes of the present invention, enhancing cellulolytic activity is determined by measuring the increase in reducing sugars or the increase in total cellobiose and glucose from hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1-50 mg of protein total/g of cellulose in PCS (pre-treated corn husks), wherein the total protein is comprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% protein of a GH61 polypeptide w/w having enhancing cellulolytic activity for 1-7 days at 50 °C compared to a control hydrolysis with equal total protein load without enhancing cellulolytic activity (1-50 mg cellulolytic protein/g cellulose in PCS). In wo curgeVq rtgfgtgpekcn, woc okuVwtc fg EGNNWENCUV™ 3o7N *Pqxqz{ogu A/S, Bagsv^rd, Denmark) in the presence of 2-3 wt% total protein Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02/095014) or 2-3 wt% total beta-glucosidase protein from Aspergillus fumigatus (produced recombinantly in Aspergillus oryzae as described in WO 2002/095014) cellulase protein load is used as a source of cellulolytic activity.

[0066] The cellulolytic enzyme composition comprises a beta-glucosidase, preferably one derived from a strain of the genus Aspergillus, such as Aspergillus oryzae, such as that disclosed in WO 2002/095014 or the fusion protein having beta-glucosidase activity disclosed in WO 2008/057637 (see SEQ ID NOs: 74 or 76); or Aspergillus fumigatus, such as one disclosed herein in SEQ ID NO: 2 in WO 2005/047499 or in SEQ ID NO: 8 set forth herein; or an Aspergillus fumigatus beta-glucosidase variant disclosed in WO 2012/044915; or a strain of the genus Penicillium, such as a strain of Penicillium brasilianum disclosed in WO 2007/019442, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei. In one embodiment, the beta-glucosidase is from an Aspergillus strain, such as an Aspergillus fumigatus strain, such as an Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 8 set forth herein), or a variant thereof, which variant comprises one or more substitutions selected from the group consisting of L89M, G91L, F100D, I140V, I186V, S283G, N456E, and F512Y; as a variant of the same with the following replacements: - F100D + S283G + N456E + F512Y; - L89M + G91L + I186V + I140V; - I186V + L89M + G91L + I140V + F100D + S283G + N456E + F512Y.

[0067] The parent beta-glucosidase has at least 60% identity, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96% , such as at least 97%, such as at least 98%, such as at least 99% identity with the mature polypeptide of SEQ ID NO: 8 set forth herein.

[0068] In case the beta-glucosidase is a beta-glucosidase variant, it has at least 60% identity, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95 %, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity but less than 100% identity with the mature polypeptide of SEQ ID NO: 8 shown in this document.

[0069] In case the cellulotic enzyme composition, comprised in an enzyme composition of the invention, comprises a GH61 polypeptide, this may be a derivative of the genus Thermoascus, such as a strain of Thermoascus aurantiacus, as described in WO 2005/ 074656 as SEQ ID NO: 2 or SEQ ID NO: 9 in this document; or a derivative of the genus Thielavia, such as a strain of Thielavia terrestris, as described in WO 2005/074647 as SEQ ID NO: 7 and SEQ ID NO: 8; or a derivative of a strain of Aspergillus, such as a strain of Aspergillus fumigatus, as described in WO 2010/138754 as SEQ ID NO: 1 and SEQ ID NO: 2; or a derivative of a derived strain of Penicillium, such as a strain of Penicillium emersonii, as disclosed in WO 2011/041397 as SEQ ID NO: 2 or SEQ ID NO: 10 set forth herein.

[0070] In a preferred embodiment, the GH61 polypeptide, such as one derived from a strain of Penicillium sp., is selected from the group consisting of: (i) a GH61 polypeptide comprising the mature polypeptide of SEQ ID NO: 10 shown in this document; (ii) a GH61 polypeptide comprising an amino acid sequence having at least 60%, such as at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 10 presented in this document.

[0071] In one embodiment, the cellulolytic enzyme composition, comprising an enzyme composition of the invention, comprises a cellobiohydrolase I (CBH I), such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as such as CBHI Cel7a disclosed in SEQ ID NO: 6 in WO 2011/057140 or in SEQ ID NO: 6 set forth herein, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei.

[0072] In a preferred embodiment, cellobiohydrolase I, such as one derived from a strain of Aspergillus fumigatus, is selected from the group consisting of: (i) a cellobiohydrolase I comprising the mature polypeptide of SEQ ID NO: 6 shown herein document; (ii) a cellobiohydrolase I comprising an amino acid sequence having at least 60%, such as at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 6 presented in this document.

[0073] In one embodiment, the cellulolytic enzyme composition, comprised in an enzyme composition of the invention, comprises a cellobiohydrolase II (CBH II), such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, as disclosed in SEQ ID NO: 7 set forth herein; or a strain of the genus Trichoderma, such as Trichoderma reesei, or a strain of the genus Thielavia, such as a strain of Thielavia terrestris, such as cellobiohydrolase II CEL6A of Thielavia terrestris.

[0074] In a preferred embodiment, cellobiohydrolase II, such as one derived from a strain of Aspergillus fumigatus, is selected from the group consisting of: (i) a cellobiohydrolase II comprising the mature polypeptide of SEQ ID NO: 7 shown herein document; (ii) a cellobiohydrolase II comprising an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 7 presented in this document.

[0075] In one embodiment the cellulolytic enzyme composition, comprised in an enzyme composition of the invention, comprises a GH61 polypeptide having enhancing cellulolytic activity and a betaglucosidase.

[0076] In one embodiment, the cellulolytic enzyme composition comprises a GH61 polypeptide with enhancing cellulolytic activity derived from a strain of Penicillium, such as a strain of Penicillium emersonii, such as that described as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 shown herein, and a beta-glucosidase.

[0077] In one embodiment the cellulolytic enzyme composition, comprised in an enzyme composition of the invention, comprises a GH61 polypeptide having enhancing cellulolytic activity, a betaglucosidase, and a CBHI.

[0078] In one embodiment, the cellulolytic enzyme composition, comprised in an enzyme composition of the invention, comprises a GH61 polypeptide having enhancing cellulolytic activity derived from a strain of Penicillium, such as a strain of Penicillium emersonii, such as that described as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 set forth herein, a beta-glucosidase, and a CBHI.

[0079] In one embodiment the cellulolytic enzyme composition, comprised in an enzyme composition of the invention, comprises a GH61 polypeptide having enhancing cellulolytic activity, a betaglucosidase, a CBHI and a CBHII.

[0080] In one embodiment, the cellulolytic enzyme composition, comprised in an enzyme composition of the invention, comprises a GH61 polypeptide having enhancing cellulolytic activity derived from a strain of Penicillium, such as a strain of Penicillium emersonii, such as that described as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 set forth herein, a beta-glucosidase, a CBHI, and a CBHII.

[0081] In one embodiment, the cellulolytic enzyme composition is a cellulolytic composition from Trichoderma reesei, further comprising a GH61A polypeptide from Thermoascus aurantiacus (SEQ ID NO: 2 in WO 2005/074656 or in SEQ ID NO: 9 set forth herein) , and Aspergillus oryzae beta-glucosidase fusion protein ( WO 2008/057637 ).

[0082] In one embodiment, the cellulolytic enzyme composition is a cellulolytic composition from Trichoderma reesei, further comprising GH61A polypeptide from Thermoascus aurantiacus with increased cellulolytic activity (SEQ ID NO: 2 in WO 2005/074656 or in SEQ ID NO: 9 shown herein) and beta-glucosidase from Aspergillus fumigatus (SEQ ID NO: 2 of WO 2005/047499 or in SEQ ID NO: 8 herein).

[0083] In another embodiment, the cellulolytic enzyme composition is a cellulolytic enzyme composition from Trichoderma reesei, further comprising a GH61A polypeptide from Penicillium emersonii disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 disclosed in this document, and Aspergillus fumigatus beta-glucosidase disclosed as SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 8 disclosed herein, or a variant thereof, which variant has one, preferably all, of the following substitutions: F100D , S283G, N456E, F512Y, and optionally CBHI from Aspergillus fumigatus, e.g. that disclosed as SEQ ID NO: 6 in WO 2011/057140 and SEQ ID NO: 6 set forth herein, and CBH II from Aspergillus fumigatus, e.g. , the one disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 presented herein.

[0084] In one embodiment the cellulolytic enzyme composition, comprised in an enzyme composition of the invention, comprises one or more of the following components (i) a cellobiohydrolase I from Aspergillus fumigatus; (ii) a cellobiohydrolase II from Aspergillus fumigatus; (iii) an Aspergillus fumigatus beta-glucosidase or a variant thereof.

[0085] In one embodiment Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 8 shown herein), comprises one or more substitutions selected from the group consisting of L89M, G91L, F100D, I140V, I186V, S283G, N456E, and F512Y ; as a variant of the same, with the following replacements: - F100D + S283G + N456E + F512Y; - L89M + G91L + I186V + I140V; or - I186V + L89M + G91L + I140V + F100D + S283G + N456E + F512Y.

[0086] In one embodiment, the cellulolytic composition further comprises the GH61 polypeptide from Penicillium sp. shown in SEQ ID NO: 10 set forth in this document; or a GH61 polypeptide comprising an amino acid sequence having at least 60%, such as at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91% , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO : 10 presented in this document.

Glucoamylase

[0087] The enzyme composition of the invention comprises a glucoamylase. Glucoamylase can be of any origin, such as fungal or bacterial origin.

[0088] In one embodiment the glucoamylase may be a derivative of a strain of Trametes, such as a strain of Trametes cingulata (SEQ ID NO: 12, shown herein); or a strain of Pachykytospora, such as a strain of Pachykytospora papyracea; or a strain of Leucopaxillus, such as a strain of Leucopaxillus giganteus (all disclosed in WO 2006/069289 ).

[0089] In a preferred embodiment, the glucoamylase comprised in an enzyme composition of the invention is derived from a strain of Trametes cingulata, such as one selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 12 shown in this document; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 12 presented in this document.

[0090] In one embodiment, the glucoamylase, comprised in an enzyme composition of the invention, is from a strain of Aspergillus, preferably Aspergillus niger, Aspergillus awamori, or Aspergillus oryzae; or a strain of Trichoderma, preferably Trichoderma reesei, or a strain of Talaromyces, preferably Talaromyces emersonii (SEQ ID NO: 11 herein). In one embodiment, the glucoamylase, such as one derived from a strain of Talaromyces emersonii, is selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 11 set forth herein; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 11 presented in this document.

[0091] In another embodiment, the glucoamylase is derived from a strain of Penicillium, such as a strain of Penicillium oxalicum.

[0092] In one embodiment, the glucoamylase, such as one derived from a strain of Penicillium oxalicum, is selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 16 set forth herein; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 16 presented in this document.

[0093] In one embodiment, the glucoamylase is derived from a strain of Gloeophyllum, such as a strain of Gloeophyllum sepiarium or Gloeophyllum trabeum, such as one disclosed in WO 2011/068803 as any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16. In a preferred embodiment, the glucoamylase is SEQ ID NO: 2 in WO 2011/068803 or SEQ ID NO: 4 shown herein. In another embodiment, the glucoamylase is SEQ ID NO: 18 in WO 2011/068803.

[0094] In a preferred embodiment, the glucoamylase, such as one derived from a strain of Gloeophyllum sepiarium, is selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 4 set forth herein; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 4 presented in this document.

[0095] In a further embodiment the glucoamylase is derived from a strain of the genus Pycnoporus, in particular a strain of Pycnoporus sanguineus, such as a strain described in WO 2011/066576 (SEQ ID NOs 2, 4 or 6). In a preferred embodiment, the glucoamylase is the one shown in SEQ ID NO: 4 in WO 2011/066576 or SEQ ID NO: 17 presented herein.

[0096] In a preferred embodiment, the glucoamylase, such as one derived from a strain of Pycnoporus sanguineus, is selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 17 set forth herein; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 17 presented in this document.

[0097] Also contemplated are glucoamylases that exhibit high sequence identity with any of the aforementioned glucoamylases, i.e., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% , at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity with any of the mature parts of the aforementioned enzyme sequences

[0098] In a preferred embodiment, the glucoamylase, such as one derived from a strain of Gloeophyllum trabeum, is selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 18 set forth herein; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 18 presented in this document.

[0099] In a preferred embodiment, the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 has one of the following substitutions: V59A; S95P; A121P; T119W; S95P+A121P; V59A+S95P; S95P+T119W; V59A+S95P+A121P; or S95P+T119W+A121P, especially S95P+A121P. In a preferred embodiment, the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 has one of the following substitutions: V59A; S95P; A121P; T119W; S95P+A121P; V59A+S95P; S95P+T119W; V59A+S95P+A121P; or S95P+T119W+A121P, especially S95P+A121P (using SEQ ID NO: 18 shown in this document for numbering). All Gloeophyllum trabeum glucoamylase variants disclosed in co-pending application EP13165995.5 and PCT/EP2014/058692 are incorporated herein by reference.

[00100] A variant comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 18 set forth herein.

alpha-amylase

[00101] The enzyme composition of the invention comprises an alpha-amylase. The alpha-amylase can be of any origin, such as fungal or bacterial origin. In a preferred embodiment, the alpha-amylase is an acidic alpha-amylase, i.e., having a pH optimum below pH 7.

[00102] In one embodiment, the alpha-amylase can be derived from a strain of the genus Rhizomucor, preferably a strain of Rhizomucor pusillus, such as that shown in SEQ ID NO: 3 in WO 2013/006756 (see, for example, Table 1 in Example 1 incorporated herein by reference), or from the genus Meripilus, preferably a strain of Meripilus giganteus.

[00103] In a preferred embodiment, the alpha-amylase is derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), disclosed as V039 in Table 5 in WO 2006/069290, or in SEQ ID NO: 13 shown in this document.

[00104] In a preferred embodiment, the alpha-amylase is derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), disclosed in WO 2013/006756 (incorporated herein by reference), or SEQ ID NO: 13 shown in this document.

[00105] In one embodiment Rhizomucor pusillus alpha-amylase or Rhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD) has at least one of the following substitutions or combinations of substitutions : D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S + Y141W; A76G + Y141W; G128D + Y141W; G128D + D143N; P219C + Y141W; N142D + D143N; Y141W + K192R; Y141W + D143N; Y141W + N383R; Y141W + P219C + A265C; Y141W + N142D + D143N; Y141W + K192R V410A; G128D + Y141W + D143N; Y141W + D143N + P219C; Y141W + D143N + K192R; G128D + D143N + K192R; Y141W + D143N + K192R + P219C; G128D + Y141W + D143N + K192R; or G128D + Y141W + D143N + K192R + P219C, especially G128D+D143N (using SEQ ID NO: 13 shown in this document for numbering).

[00106] In one embodiment, the Rhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), is selected from the group consisting of: (i) an alpha-amylase comprising the mature polypeptide of SEQ ID NO: 13 disclosed herein; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 13 presented in this document.

[00107] In a preferred embodiment, the alpha-amylase is a variant of alpha-amylase from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), wherein the alpha-amylase variant comprises a amino acid sequence having at least 60%, at least 70%, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity but less than 100% for the mature polypeptide of SEQ ID NO: 13 presented in this document.

[00108] In a preferred embodiment, the alpha-amylase variant has one of the aforementioned substitutions, such as: G128D, Y141W, D143W or K192R.

[00109] In a preferred embodiment, alpha-amylase (using SEQ ID NO: 13, shown herein for numbering) has one of the following substitutions: Y141W+D143N.

[00110] In a preferred embodiment, alpha-amylase has the following substitutions: G128D+Y141W+D143N.

[00111] In a preferred embodiment, alpha-amylase has the following substitutions: G128D+Y141W+D143N+K192R;

[00112] In a preferred embodiment, alpha-amylase has the following substitutions: G128D+D143N.

[00113] A variant comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 13 set forth herein.

protease

[00114] An enzyme composition of the invention may optionally comprise a protease. The protease can be of any origin, such as fungal or bacterial origin. In a preferred embodiment, the protease is a metalloprotease. In another preferred embodiment, the protease is a serine protease.

[00115] In one embodiment, the protease is of fungal origin.

[00116] In one embodiment the protease, optionally comprised in an enzyme composition of the invention, is a metalloprotease derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670, such as the metalloprotease disclosed as the mature part of SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature polypeptide of SEQ ID NO: 3 disclosed herein.

[00117] In one embodiment, the protease, such as one derived from a strain of Thermoascus aurantiacus, is selected from the group consisting of: (i) a protease comprising the mature polypeptide of SEQ ID NO: 3 set forth herein; (ii) a protease comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 3 presented in this document.

[00118] In one embodiment, the protease is of bacterial origin.

[00119] In one embodiment, the protease, optionally comprised in an enzyme composition of the invention, is derived from a Pyrococcus strain, such as a Pyrococcus furiosus strain, such as the protease shown in SEQ ID NO: 1 in US 6,358,726 or SEQ ID NO: 5 shown in this document.

[00120] In one embodiment, the protease, such as one derived from Pyrococcus furiosus, is selected from the group consisting of: (i) a protease comprising the mature polypeptide of SEQ ID NO: 5 set forth herein; (ii) a protease comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 5 presented in this document.

[00121] In another preferred embodiment, the protease optionally comprised in an enzyme composition of the invention is a serine protease, such as a protease from the S53 family of peptidases. The serine proteases of the S53 family of peptidases comprise two different types of peptidases: tripeptidyl aminopeptidases (exo-type) and endopeptidases; as described in 1993, Biochem. J. 290:205-218 and in the MEROPS protease database, release, 9.4 (31 January 2011) (www.merops.ac.uk). The database is described in Rawlings, N.D., Barrett, A.J. and Bateman, A., 2010, "MEROPS: the peptidase database", Nucl. Res. 38: D227-D233. In a preferred embodiment, the protease is a protease from the S53 family of peptidases derived from a strain of Meripilus, preferably a strain of Meripilus giganteus. In one embodiment, the protease is the mature sequence of Meripilus giganteus protease 3 (protease of the S53 family of peptidases) discussed in Example 2 in WO 2014/037438 and shown as SEQ ID NO: 20 herein. In one embodiment, the protease is the mature sequence of Meripilus giganteus protease 3 shown as SEQ ID NO: 19 herein and SEQ ID No: 5 in WO 2014/037438. In a preferred embodiment, the protease, such as Meripilus giganteus protease 3, is selected from the group consisting of: (i) a protease comprising the mature polypeptide of SEQ ID NO: 19 set forth herein; (ii) a protease comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 19 presented in this document.

[00122] In one embodiment, the protease, such as Meripilus giganteus protease 3, is selected from the group consisting of: (i) a protease comprising the mature polypeptide of SEQ ID NO: 20 set forth herein; (ii) a protease comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 20 presented in this document.

pullulanase

[00123] An enzyme composition of the invention may optionally comprise a pullulanase. The pullulanase can be of any origin, such as fungal or bacterial origin.

[00124] In one embodiment, the pullulanase, optionally comprised in an enzyme composition of the invention, is derived from a strain of Bacillus sp, such as that shown in SEQ ID NO: 15 set forth herein, or a strain of Bacillus demificans.

[00125] In one embodiment, the pullulanase, such as one derived from Bacillus sp, is selected from the group consisting of: (i) a pullulanase comprising the mature polypeptide of SEQ ID NO: 15 set forth herein; (ii) a pullulanase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 15 presented in this document.

[00126] In one embodiment the enzyme composition of the invention further comprises a pullulanase, such as pullulanase from Bacillus sp. and a glucoamylase from Talaromyces emersonii and/or glucoamylase from Gloeophyllum sepiarium.

trehalase

[00127] According to the invention, the enzyme composition may further comprise a trehalase. The trehalase can be of any origin, such as fungal or bacterial origin.

[00128] In one embodiment, the trehalase is of fungal origin, such as derived from a strain of Trichoderma, such as Trichoderma reesei, such as shown in SEQ ID NO: 14 herein.

[00129] In one embodiment, the trehalase, such as one derived from Trichoderma reesei, is selected from the group consisting of: (i) a trehalase comprising the mature polypeptide of SEQ ID NO: 14 set forth herein; (ii) a trehalase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 14 presented in this document.

pectinase

[00130] According to the invention, the enzyme composition may further comprise a pectinase, such as a pectin lyase (also known as pectolyase) and/or a polygalacturonase, or a combination thereof.

[00131] The pectinase can be of any origin, such as fungal or bacterial origin.

[00132] In a preferred embodiment, the pectinase is a pectin lyase (EC 4.2.2.10).

[00133] In one embodiment, the pectin lyase is derived from a strain of Aspergillus, such as Aspergillus niger.

[00134] In a preferred embodiment, the pectinase is a polygalacturonase (EC. 3.2.1.15).

[00135] In one embodiment, the polygalacturonase is derived from a strain of Aspergillus, such as Aspergillus aculeatus.

[00136] In one embodiment, the pectinase is a combination of pectin lyase and polygalacturonase. In one embodiment, the pectinase is a combination of pectin lyase derived from Aspergillus niger and polygalacturonase derived from Aspergillus aculeatus. Enzyme composition modalities

[00137] In one embodiment the enzyme composition of the invention comprises a fungal glucoamylase and a fungal alpha-amylase, and optionally a protease.

[00138] In a preferred embodiment, the enzyme composition comprises glucoamylase from Gloeophyllum trabeum shown in SEQ ID NO: 18 presented herein having one or more of the following substitutions: S95P, A121P, preferably S95P+A121P; and an alpha-amylase, preferably a Rhizomucor pusillus-derived alpha-amylase with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), shown in SEQ ID NO: 13 set forth herein, having one or more of the following substitutions: G128D, D143N, preferably G128D+D143N.

[00139] In another preferred embodiment, the enzyme composition comprises the Pycnoporus sanguineus glucoamylase shown in SEQ ID NO: 17 presented herein; and an alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably as shown in SEQ ID NO: 13 herein, preferably having one or more of the following replacements: G128D, D143N, especially G128D+D143N.

[00140] In a preferred embodiment, the enzyme composition comprises glucoamylase from Gloeophyllum trabeum shown in SEQ ID NO: 4 presented herein, and an alpha-amylase, preferably an alpha-amylase derived from Rhizomucor pusillus with a glucoamylase linker from Aspergillus niger and starch binding domain (SBD), shown in SEQ ID NO: 13 shown herein, having one or more of the following substitutions: G128D, D143N, preferably G128D+D143N.

[00141] In a preferred embodiment, the enzyme composition comprises the glucoamylase from Trametes cingulata shown in SEQ ID NO: 11 presented in this document, and an alpha-amylase, preferably an alpha-amylase derived from Rhizomucor pusillus with a glucoamylase linker from Aspergillus niger and starch binding domain (SBD), shown in SEQ ID NO: 13 shown herein, having one or more of the following substitutions: G128D, D143N, preferably G128D+D143N.

[00142] In one embodiment, the enzyme composition comprises i) fungal glucoamylase; ii) fungal alpha-amylase; iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide, beta-glucosidase, CBH I and CBH II; iv) optionally a protease.

[00143] In one embodiment, the enzyme composition of the invention comprises i) glucoamylase from Trametes cingulata; ii) Rhizomucor pusillus alpha-amylase, or a variant thereof; iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide from Penicillium emersonii, beta-glucosidase from Aspergillus fumigatus with the following substitutions: F100D, S283G, N456E, F512Y, and optionally CBH I from Aspergillus fumigatus and CBH II from Aspergillus fumigatus; iv) optionally, a protease from Thermoascus aurantiacus, or a variant thereof.

[00144] In one embodiment, the enzyme composition of the invention comprises i) glucoamylase from Trametes cingulata; ii) Rhizomucor pusillus alpha-amylase, or a variant thereof; iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide from Penicillium emersonii, beta-glucosidase from Aspergillus fumigatus with the following substitutions: F100D, S283G, N456E, F512Y (using SEQ ID NO: 8 shown in this document for numbering), and optionally Aspergillus fumigatus CBH I and Aspergillus fumigatus CBH II; iv) optionally, a protease from Pyropoccus furiosus, preferably as shown in.

[00145] In one embodiment, the enzyme composition of the invention comprises i) glucoamylase derived from Trametes cingulata; ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), or a variant thereof; iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei. iv) optionally, a protease from Thermoascus aurantiacus, or a variant thereof and/or Pyrococcus furiosus, or Meripilus giganteus.

[00146] In one embodiment, the enzyme composition comprises i) fungal glucoamylase; ii) fungal alpha-amylase; iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide, CBH I and CBH II beta-glucosidase; iv) pectinase, preferably a pectin lyase or a polygalacturonase, or a combination thereof.

[00147] In one embodiment, the pectinase is a combination of pectin lyase derived from Aspergillus niger and polygalacturonase derived from Aspergillus aculeatus.

[00148] In one embodiment, the pectinase is a combination of pectin lyase and polygalacturonase. In one embodiment, the pectinase is a combination of pectin lyase derived from Aspergillus niger and polygalacturonase derived from Aspergillus aculeatus.

[00149] In one embodiment, the enzyme composition comprises i) fungal glucoamylase; ii) fungal alpha-amylase; iii) pectinase, preferably a pectin lyase or a polygalacturonase, or a combination thereof. iv) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide, CBH I and CBH II beta-glucosidase; v) protease.

[00150] In one embodiment, the enzyme composition comprises i) fungal glucoamylase; ii) fungal alpha-amylase; iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide, CBH I and CBH II beta-glucosidase; iv) optionally a protease.

[00151] In one embodiment, the enzyme composition of the invention comprises i) glucoamylase from Trametes cingulata; ii) Rhizomucor pusillus alpha-amylase, or a variant thereof; iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide from Penicillium emersonii, beta-glucosidase from Aspergillus fumigatus with the following substitutions: F100D, S283G, N456E, F512Y, and optionally CBH I from Aspergillus fumigatus and CBH II from Aspergillus fumigatus; iv) pectin lyase derived from Aspergillus niger or polygalacturonase derived from Aspergillus aculeatus, or a combination thereof; v) ) protease from Thermoascus aurantiacus, or a variant thereof, or Pyrococcus furiosus, or Meripilus giganteus.

[00152] In a preferred embodiment, the enzyme composition comprises i) Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 herein having one or more of the following substitutions: S95P+A121P; ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), shown in SEQ ID NO: 13 shown herein, having the following substitutions: G128D+D143N; iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide from Penicillium emersonii, beta-glucosidase from Aspergillus fumigatus with the following substitutions: F100D, S283G, N456E, F512Y, and optionally CBH I from Aspergillus fumigatus and CBH II from Aspergillus fumigatus; optionally iv) Thermoascus aurantiacus protease, or a variant thereof.

[00153] In a preferred embodiment, the enzyme composition comprises i) glucoamylase from Pycnoporus sanguineus shown in SEQ ID NO: 17 presented herein; ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), shown in SEQ ID NO: 13 shown herein, having the following substitutions: G128D+D143N; iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide from Penicillium emersonii, beta-glucosidase from Aspergillus fumigatus with the following substitutions: F100D, S283G, N456E, F512Y, and optionally CBH I from Aspergillus fumigatus and CBH II from Aspergillus fumigatus; optionally iv) Thermoascus aurantiacus protease, or a variant thereof.

[00154] In a preferred embodiment, the enzyme composition comprises i) glucoamylase from Pycnoporus sanguineus shown in SEQ ID NO: 17 presented herein; ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), shown in SEQ ID NO: 13 shown herein, having the following substitutions: G128D+D143N; iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide from Penicillium emersonii, beta-glucosidase from Aspergillus fumigatus with the following substitutions: F100D, S283G, N456E, F512Y, and optionally CBH I from Aspergillus fumigatus and CBH II from Aspergillus fumigatus; optionally iv) Meripipus giganteus protease, such as Meripipus giganteus protease 3.

[00155] In a preferred embodiment, the enzyme composition comprises i) glucoamylase from Pycnoporus sanguineus shown in SEQ ID NO: 17 shown herein, or a glucoamylase having at least 60%, at least 70%, e.g., at least at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17 shown herein; ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), shown in SEQ ID NO: 13 shown herein, or an alpha-amylase having at least 60%, at least at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13 shown herein, preferably having the following substitutions: G128D+D143N; iii) optionally a cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide from Penicillium emersonii, beta-glucosidase from Aspergillus fumigatus with the following substitutions: F100D, S283G, N456E, F512Y (sing SEQ ID NO: 8 presented in this document for numbering), and optionally CBH I from Aspergillus fumigatus and CBH II from Aspergillus fumigatus; iv) Meripipus giganteus protease, such as Meripipus giganteus protease 3, or a protease having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of sequence identity with SEQ ID NO: 20 shown in this document.

[00156] In a preferred embodiment, the enzyme composition comprises i) glucoamylase from Gloeophyllum sepiarium shown in SEQ ID NO: 4 shown herein, or a glucoamylase having at least 60%, at least 70%, e.g., at least at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 4 shown herein; ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), shown in SEQ ID NO: 13 shown herein, or an alpha-amylase having at least 60%, at least at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13 shown herein, preferably having the following substitutions: G128D+D143N; iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, preferably further comprising a GH61 polypeptide from Penicillium emersonii, beta-glucosidase from Aspergillus fumigatus with the following substitutions: F100D, S283G, N456E, F512Y (using SEQ ID NO: 8 for numbering), and optionally Aspergillus fumigatus CBH I and Aspergillus fumigatus CBH II; optionally iv) protease from Thermoascus aurantiacus, shown in SEQ ID NO: 3 set forth herein, or a protease having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least at least 99% sequence identity with SEQ ID NO: 3 shown herein, or a variant thereof.

[00157] In a preferred embodiment, the enzyme composition comprises i) glucoamylase from Trametes cingulata shown in SEQ ID NO: 12 herein, or a glucoamylase having at least 60%, at least 70%, e.g., at least at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 12 shown herein; ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), shown in SEQ ID NO: 13 shown herein, or an alpha-amylase having at least 60%, at least at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13 shown herein, having the following substitutions: G128D+D143N; iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, preferably further comprising a GH61 polypeptide from Penicillium emersonii, beta-glucosidase from Aspergillus fumigatus with the following substitutions: F100D, S283G, N456E, F512Y (using SEQ ID NO: 8 presented in this document for numbering), and optionally CBH I from Aspergillus fumigatus and CBH II from Aspergillus fumigatus; optionally iv) Thermoascus aurantiacus protease, or a protease having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3 shown in this document, or a variant thereof.

Invention Process

[00158] As mentioned above an enzyme composition of the invention can be suitably used in a crude starch hydrolysis (RSH) process for producing desired sugars and fermentation products. In RSH processes, the starch does not gelatinize when the process is carried out at temperatures below the gelatinization temperature of the initial starch in question.

[00159] The desired fermentation product may, in one embodiment, be ethanol produced from ungelatinized (i.e., uncooked), preferably ground grains, such as corn, or small grains, such as wheat, oats, barley, rye, rice or cereals such as sorghum. Examples of suitable starch-containing starting materials are listed in the "Starch-Containing Materials" section below.

[00160] Accordingly, in that aspect, the invention relates to processes for producing fermentation products from starch-containing material comprising: (i) saccharification of a starch-containing material at a temperature below the initial gelatinization temperature; and (ii) fermentation using a fermentation organism; wherein the saccharification and/or fermentation is carried out in the presence of the following enzymes: glucoamylase and alpha-amylase, and optionally a protease.

[00161] In one embodiment, the glucoamylase is the glucoamylase from Gloeophyllum trabeum shown in SEQ ID NO: 18 presented herein having one or more of the following substitutions: S95P, A121P, preferably S95P+A121P, and alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), shown in SEQ ID NO: 13 shown herein, having one or more of the following substitutions: G128D, D143N, preferably G128D+D143N.

[00162] In a preferred embodiment, the invention relates to processes for producing fermentation products from starch-containing material comprising: (a) saccharification of a starch-containing material at a temperature below the initial gelatinization temperature; and (b) fermentation using a fermentation organism; wherein the saccharification and/or fermentation is carried out in the presence of the following enzymes: i) glucoamylase; ii) alpha-amylase; iii) cellulotic enzyme composition; iv) optionally protease.

[00163] In a preferred embodiment, the enzyme can be added as an enzyme composition of the invention. In a preferred embodiment, steps (a) and (b) are carried out simultaneously (ie, single-step fermentation). However, step (a) and (b) can also be performed sequentially. In accordance with this aspect of the invention, the desired fermentation product, such as ethanol, can be produced without liquefying the aqueous slurry containing the starch-containing material. The process of the invention includes saccharifying the starch-containing material (e.g., bleached), e.g., granular starch, below the initial gelatinization temperature, preferably in the presence of an enzyme composition of the invention. The sugars generated during saccharification can be simultaneously fermented into the desired fermentation product by the appropriate fermenting organism(s).

[00164] In a preferred embodiment, a cellulolytic enzyme composition is one described above in the "Cellulolytic enzyme composition" section. Preferably, the cellulolytic enzyme composition is added to the process of the invention.

[00165] In a preferred embodiment, the cellulolytic enzyme composition is comprised in an enzyme composition of the invention. According to the invention, enzymes, preferably in the form of an enzyme composition of the invention, are added to saccharification and/or fermentation, preferably simultaneous saccharification and fermentation. It should be understood that the enzymes can also be added individually or as a composition of two, three, four or more enzymes. In one embodiment the glucoamylase and alpha-amylase are added as a blend composition and the cellulolytic enzyme composition and optional protease are added separately. In another embodiment the cellulolytic enzyme composition, the glucoamylase, and the alpha-amylase are added as an enzyme composition and the optional protease is added separately. All enzymes may be added as an enzyme composition comprising a glucoamylase, an alpha-amylase, optionally a cellulolytic enzyme composition, and/or a protease, and optionally other enzymes including pullulanase and/or pectinase such as pectin lyase or polygalacturonase.

[00166] The fermentation product, such as especially a liquid fermentation product, such as ethanol, can optionally be recovered after fermentation, eg by distillation. Subsequent to fermentation, the fermentation product can be separated from the fermentation medium. The fermentation medium can be distilled to extract the desired fermentation product or the desired fermentation product can be extracted from the fermentation medium by microfiltration or membrane filtration techniques. Alternatively, the fermentation product can be recovered by separation. Methods for recovering fermentation products are well known in the art.

[00167] An example of a fermenting organism is yeast, preferably a strain of Saccharomyces cerevisiae. According to the invention, Saccharomyces cerevisiae can be used for the production of ethanol. Other suitable fermenting organisms are listed in the "Fermenting Organisms" section below.

[00168] The term "initial gelatinization temperature" means the lowest temperature at which starch gelatinization begins. In general, starch heated in water begins to gelatinize between about 50°C and 75°C. The exact gelatinization temperature depends on the specific starch and depends on the degree of cross-linking of the amylopectin. The initial gelatinization temperature can be readily determined by those skilled in the art. The initial gelatinization temperature can vary according to the plant species, the particular variety of plant species as well as the growing conditions. In the context of the present invention, the initial gelatinization temperature of a given starch-containing material can be determined as the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein. S. and Lii. C., Starch/Starke, Vol. 44 (12) pg. 461-466 (1992).

[00169] Prior to step (a) an aqueous slurry of starch-containing material, such as granular starch, may be prepared having 10-55% by weight of dry solids, preferably 25-45% by weight of dry solids, more preferably 30 -40% dry solids of starch-containing material. The slurry may include water and/or process waters such as vinasse (countercurrent), scrubber water, evaporator condensate or distillate, water discharged from distillation sides, or process water from other fermentation products facilities. Because the process of the invention is carried out below the initial gelatinization temperature and therefore no significant increase in viscosity takes place, if desired, high levels of vinasse can be used. In one embodiment, the aqueous slurry contains from about 1 to about 70% by vol., preferably 15-60% by vol., especially from about 30 to 50% by vol. of water and/or process waters, such as vinasse (countercurrent), scrubber water, evaporator condensate or distillate, water discharged from distillation sides, or process water from other fermentation products facilities, or combinations thereof, or similar.

[00170] In one embodiment, countercurrent or other recycled flow is added to the suspension before step (a), or saccharification (step (a)), or simultaneous saccharification and fermentation steps (step (a) and step ( b) combined).

[00171] After being subjected to a process of the invention, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or preferably at least 99% of the dry solids in the starch-containing material are converted to a hydrolyzate of soluble starch.

[00172] A process of the invention is carried out at a temperature below the initial gelatinization temperature, which means that the temperature at which a separate step (a) is carried out typically lies in the range between 25-75 °C, such such as between 30-70 °C, or between 45-60 °C.

[00173] In a preferred embodiment, the temperature during the fermentation in step (b) or the simultaneous saccharification and fermentation in steps (a) and (b) is between 25 °C and 40 °C, preferably between 28 °C and 36°C, such as between 28°C and 35°C, such as between 28°C and 34°C, such as about 32°C.

[00174] In one embodiment of the invention, fermentation is carried out for 30 to 150 hours, preferably 48 to 96 hours. 66.

[00175] In one embodiment, the fermentation is carried out such that the sugar level, such as glucose level, is maintained at a low level such as below 6% by weight, such as below about 3% by weight. weight, such as below about 2% by weight, such as below about 1% by weight, such as below about 0.5% by weight, such as below about 0.25% by weight, such as below about 0.1% by weight. Such low sugar levels can be achieved by simply employing adjusted amounts of enzymes and fermenting organism. A person skilled in the art can easily determine which doses/amounts of enzyme and fermenting organism to use. The amounts of enzyme and fermenting organism employed can also be selected to maintain low concentrations of maltose in the fermentation broth. For example, the level of maltose can be maintained below about 0.5% by weight, such as below about 0.2% by weight.

[00176] The process of the invention can be carried out at a pH of 3 to 7, preferably 3 to 6 or more preferably 3.5 to 5.0.

[00177] The term "granular starch" means uncooked raw starch, i.e. starch in its natural form found in, for example, cereals, tubers or grains. Starch is formed within plant cells as tiny, water-insoluble granules. When placed in cold water, the starch granules can absorb a small amount of liquid and swell. At temperatures up to about 50°C to 75°C, the swelling may be reversible. However, at higher temperatures, an irreversible swelling called "gelatinization" begins. The granular starch can be a highly refined starch, preferably at least 90%, at least 95%, at least 97% or at least 99.5% pure, or it can be a more crude starch-containing material comprising whole grains (e.g. g. milled) including non-starch fractions such as germination residues and fibers.

[00178] The raw material, such as whole grains, can be reduced in particle size, eg by milling, in order to open the structure and allow further processing. Examples of suitable particle sizes are described in US4514496 and WO2004/081193 (incorporated by reference). Two processes are preferred according to the invention: dry and wet milling. In dry milling, whole grains are ground and used. Wet milling gives good separation of germ and flour (starch and protein granules) and is often applied in locations where starch hydrolyzate is used in the production of eg syrups. Both dry and wet milling are well known in the starch processing art.

[00179] In one embodiment, the particle size is reduced to between 0.05 to 3.0 mm, preferably 0.1-0.5 mm, or such that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material fits through a screen having a grid of 0.05 to 3.0 mm, preferably a grid of 0.1-0.5 mm. In a preferred embodiment the starch-containing material is prepared by reducing the particle size of the starch-containing material, preferably by milling, such that at least 50% of the starch-containing material has a particle size of 0.1- 0.5 mm.

[00180] According to the invention, the enzymes or the enzyme composition is added such that the glucoamylase is present in an amount of 0.001 to 10 AGU/g DS preferably 0.01 to 5 AGU/g DS , especially 0.1 to 0.5 AGU/g DS.

[00181] According to the invention, the enzymes or the enzyme composition is added such that the alpha-amylase is present or is added in an amount of 0.001 to 10 AFAU/g DS, preferably 0.01 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS or 0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS.

[00182] According to the invention, the enzymes or the enzyme composition is added such that the cellulolytic enzyme composition is present or is added in an amount of 1-10,000 micrograms of PE/g of DS, such as 2- 5,000, such as 3 and 1,000, such as 4 and 500 microgram PE/g DS.

[00183] According to the invention, the enzymes or the enzyme composition is added such that the cellulolytic enzyme composition is present or is added in an amount in the range of 0.1-100 FPU per gram of total solids (TS ), preferably 0.5-50 FPU per gram TS, especially 1-20 FPU per gram TS.

[00184] In one embodiment of the invention, the enzymes or enzyme composition is added such that the protease is present in an amount of 0.0001-1 mg of enzyme protein per g of DS, preferably 0.001 to 0.1 mg of enzyme protein per g of DS. Alternatively, the protease is present in an amount of 0.0001 to 1 LAPU/g DS, preferably 0.001 to 0.1 LPAU/g DS, and/or 0.0001 to 1 mAU-RH/g DS, preferably 0.001 to 0.1 mAU-RH/g DS.

[00185] In one embodiment of the invention, the enzymes or enzyme composition is added such that the protease is present or is added in an amount in the range of 1-1000 µg PE/g DS, such as 2-500 µg PE/g DS, such as 3-250 µg PE/g DS.

[00186] In a preferred embodiment, the ratio of glucoamylase to alpha-amylase is between 99:1 and 1:2, such as between 98:2 and 1:1, such as between 97:3 and 2:1, such as between 96:4 and 3:1, such as 97:3, 96:4, 95:5, 94:6, 93:7, 90:10, 85:15, 83:17 or 65:35 (mg of PE glucoamylase: mg of PE alpha amylase).

[00187] In a preferred embodiment, the total dose of glucoamylase and alpha-amylase according to the invention is 10-1,000 μg/g DS, such as 50-500 μg/g DS, such as 75- 250 µg/g DS.

[00188] In a preferred embodiment, the total dose of cellulolytic enzyme composition added is 10-500 µg/g DS, such as 20400 µg/g DS, such as 20-300 µg/g DS.

[00189] In one embodiment, the dose of protease added is 1-200 µg/g DS, such as 2-100 µg/g DS, such as 3-50 µg/g DS.

Starch Containing Materials

[00190] According to the process of the invention, any suitable starch-containing starting material can be used, including granular starch (raw uncooked starch). Starting material is generally selected based on the desired fermentation product. Examples of starch-containing starting materials suitable for use in processes of the present invention include cereals, tubers or grains. Specifically, the starch-containing material can be corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, beans, or sweet potatoes, or mixtures thereof. Both waxy and non-waxy types of corn and barley are contemplated.

[00191] In a preferred embodiment, the starch-containing starting material is corn.

[00192] In a preferred embodiment, the starch-containing starting material is wheat.

[00193] In a preferred embodiment, the starch-containing starting material is barley.

[00194] In a preferred embodiment, the starch-containing starting material is rye.

[00195] In a preferred embodiment, the starch-containing starting material is milo.

[00196] In a preferred embodiment, the starch-containing starting material is sago.

[00197] In a preferred embodiment, the starch-containing starting material is cassava.

[00198] In a preferred embodiment, the starch-containing starting material is tapioca.

[00199] In a preferred embodiment, the starch-containing starting material is sorghum.

[00200] In a preferred embodiment, the starch-containing starting material is rice.

[00201] In a preferred embodiment, the starch-containing starting material is peas.

[00202] In a preferred embodiment, the starch-containing starting material is beans.

[00203] In a preferred embodiment, the starch-containing starting material is sweet potato.

Fermenting Organisms

[00204] According to the invention, "fermenting organism" refers to any organism, including bacterial and fungal organisms, suitable for use in a fermentation process and capable of producing a desired fermentation product. Especially suitable fermenting organisms are capable of fermenting, i.e. converting, sugars, such as glucose or maltose, directly or indirectly into the desired fermentation product, especially ethanol. Examples of fermenting organisms include fungal organisms such as yeast. Preferred yeasts include strains of Saccharomyces spp., in particular Saccharomyces cerevisiae.

[00205] Fermenting organisms engineered to produce one or more enzymes, such as a saccharolytic enzyme, such as glucoamylase, are also contemplated. Contemplated yeast, especially Saccharomyces cerevisiae, can also be manipulated to produce less glycerol. Examples of such yeasts can be found in WO/2011/153516 (Mascoma).

[00206] In one embodiment, the fermenting organism is added to the fermentation medium such that the count of viable fermenting organism, such as yeast, per ml of fermentation medium is in the range of 105 to 1012, preferably 107 to 1010, especially about 5x107.

[00207] Yeasts available on the market include, for example, the yeast TGF UVAT™ g GVJCPQN TGF (disponibüizcda rgnc HgtogpVkulNguchhtg. GWA+, HCLK *fkurqpkdknkzcfc rgnc Hlgiuelimcnifu [gcuV. GWC+. , USA), BIOFERM AFT and XR (available from NABC - North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).

Fermentation Products

[00208] The term "fermentation product" means a product produced by a process including a fermentation step of the invention using one or more fermenting organisms. Fermentation products contemplated in accordance with the invention include alcohols (e.g., ethanol, methanol, butanol); organic acids (eg, citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (eg, glutamic acid); gases (eg, H2 and CO2); antibiotics (eg, penicillin and tetracycline); enzymes; vitamins (eg, riboflavin, B12, beta-carotene); and hormones. In a preferred aspect, the fermentation product is ethanol, e.g. fuel ethanol; potable ethanol, i.e. potable neutral alcoholic beverages; or industrial ethanol or products used in the consumer alcohol industry (eg, beer and wine), the dairy industry (eg, fermented milk products), the fur industry, and the tobacco industry. Preferred types of beer include ales, stouts, porters, lagers, bitters, malts, happoushu, high alcohol beer, low alcohol beer, low calorie beer or non-alcoholic beer. The fermentation product, such as ethanol, obtained according to the invention can be preferably used as fuel. However, in the case of ethanol, it can also be used as potable ethanol.

Fermentation Medium

[00209] The term "fermentation medium" refers to the environment in which fermentation is carried out and which includes the fermentable substrate, i.e. a source of carbohydrates (e.g. glucose) that can be metabolized by the fermenting organism(s).

[00210] The fermentation medium may comprise nutrients and/or growth stimulator(s) for the fermenting organism(s). Nutrients and growth enhancers are widely used in fermentation technique and include nitrogen sources such as ammonia; vitamins; and minerals, or combinations thereof.

Examples of Invention Processes

[00211] In a preferred embodiment, the process of producing a fermentation product from the starch-containing material of the invention, comprising: (a) saccharification of a starch-containing material at a temperature below the initial gelatinization temperature; and (b) fermentation using a fermentation organism; wherein the saccharification and/or fermentation is carried out in the presence of the following enzymes: i) glucoamylase derived from Trametes cingulata, Gloeophyllum trabeum, Gloeophyllum sepiarium, or Pycnoporus sanguineus; ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), or a variant thereof; iii) cellulolytic enzyme composition derived from Trichoderma reesei; iv) optionally, a protease from Thermoascus aurantiacus, or a variant thereof and/or Pyrococcus furiosus, or Meripilus giganteus.

[00212] In a preferred embodiment, the process of producing a fermentation product from the starch-containing material of the invention, comprising: (a) saccharification of a starch-containing material at a temperature below the initial gelatinization temperature; and (b) fermentation using a fermentation organism; wherein saccharification and/or fermentation is carried out in the presence of the following enzymes: i) glucoamylase derived from Gloeophyllum trabeum disclosed in SEQ ID NO: 18 set forth herein, or a glucoamylase having at least 60%, at least 70%, p .eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 18 shown herein, preferably with the following substitutions: S95P+A121P; ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), or a variant thereof, shown in SEQ ID NO: 13 shown herein, or an alpha-amylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13 shown herein, preferably with the following substitutions : G128D+D143N; iii) cellulolytic enzyme composition derived from Trichoderma reesei; iv) optionally a protease from Thermoascus aurantiacus, or a protease having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3 shown in this document, or a variant thereof.

[00213] In a preferred embodiment, the process of producing a fermentation product, such as ethanol, from the starch-containing material of the invention, comprising: (a) saccharification of a starch-containing material at a temperature below the gelatinization temperature initial; and (b) fermentation using a fermentation organism; wherein saccharification and/or fermentation is carried out in the presence of the following enzymes: i) glucoamylase derived from Gloeophyllum trabeum disclosed in SEQ ID NO: 18 set forth herein, or a glucoamylase having at least 60%, at least 70%, p .eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 18 shown herein, preferably with the following substitutions: S95P+A121P; ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), or a variant thereof, shown in SEQ ID NO: 13 shown herein, or an alpha-amylase having at least 60%, at least 70%, e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13 shown herein, with the following substitutions: G128D+D143N; iii) optionally a cellulolytic enzyme composition, such as one derived from Trichoderma reesei; iv) a protease from Meripipus giganteus, such as protease 3 from Meripipus giganteus, or a protease having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 20 shown in this document.

[00214] In a preferred embodiment, the process of producing a fermentation product, such as ethanol, from the starch-containing material of the invention, comprising: (a) saccharification of a starch-containing material at a temperature below the gelatinization temperature initial; and (b) fermentation using a fermentation organism; wherein saccharification and/or fermentation is carried out in the presence of the following enzymes: i) glucoamylase derived from Gloeophyllum trabeum disclosed in SEQ ID NO: 18 set forth herein, or a glucoamylase having at least 60%, at least 70%, p .eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 18 shown herein, with the following substitutions: S95P+A121P; ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), or a variant thereof, shown in SEQ ID NO: 13 shown herein, or an alpha-amylase having at least 60%, at least 70%, e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13 shown herein, with the following substitutions: G128D+D143N; iii) cellulolytic enzyme composition derived from Trichoderma reesei; iv) a protease from Meripilus giganteus, such as protease 3 from Meripilus giganteus, such as one described in SEQ ID NO: 19 shown herein, or in SEQ ID NO: 20 shown herein, or a protease having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 19 shown in this document, or with SEQ ID NO: 20 shown in this document document, respectively.

[00215] In a preferred embodiment, the process of producing a fermentation product from the starch-containing material of the invention, comprising: (a) saccharification of a starch-containing material at a temperature below the initial gelatinization temperature; and (b) fermentation using a fermentation organism; wherein the saccharification and/or fermentation is carried out in the presence of the following enzymes: i) glucoamylase derived from Pycnoporus sanguineus shown in SEQ ID NO: 17 shown herein; or a glucoamylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at ii ) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), or a variant thereof, shown in SEQ ID NO: 13 shown herein, or an alpha-amylase having at least at least 60%, at least 70%, e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13 shown herein, preferably with the following substitutions: G128D+D143N; iii) cellulolytic enzyme composition derived from Trichoderma reesei; iv) optionally, a protease from Thermoascus aurantiacus, or a variant thereof.

[00216] In a preferred embodiment, the process of producing a fermentation product from the starch-containing material of the invention, comprising: (a) saccharification of a starch-containing material at a temperature below the initial gelatinization temperature; and (b) fermentation using a fermentation organism; wherein the saccharification and/or fermentation is carried out in the presence of the following enzymes: i) glucoamylase derived from Gloeophyllum sepiarium shown in SEQ ID NO: 4 shown herein; or a glucoamylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at ii ) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), or a variant thereof, shown in SEQ ID NO: 13 shown herein, or an alpha-amylase having at least at least 60%, at least 70%, e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13 shown herein, with the following substitutions: G128D +D143N; iii) cellulolytic enzyme composition derived from Trichoderma reesei; iv) optionally a protease from Thermoascus aurantiacus, or a protease having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3 shown in this document, or a variant thereof.

[00217] In a preferred embodiment, the process of producing a fermentation product from the starch-containing material of the invention, comprising: (a) saccharification of a starch-containing material at a temperature below the initial gelatinization temperature; and (b) fermentation using a fermentation organism; wherein the saccharification and/or fermentation is carried out in the presence of the following enzymes: i) glucoamylase derived from Trametes cingulata shown in SEQ ID NO: 12 presented herein; or a glucoamylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at ii ) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), or a variant thereof, shown in SEQ ID NO: 13 shown herein, or an alpha-amylase having at least at least 60%, at least 70%, e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13 shown herein, preferably with the following substitutions: G128D+D143N; iii) cellulolytic enzyme composition derived from Trichoderma reesei; iv) optionally a protease from Thermoascus aurantiacus, or a protease having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3 shown in this document, or a variant thereof.

MATERIALS & METHODS Materials:

[00218] GtAMG: Glucoamylase derived from Gloeophyllum trabeum disclosed in SEQ ID NO: 18 presented in this document, with the following substitutions: S95P+A121P.

[00219] PsAMG: Glucoamylase derived from Pycnoporus sanguineus disclosed as shown in SEQ ID NO: 4 in WO 2011/066576 and in SEQ ID NO: 17 presented herein.

[00220] TcAMG: Glucoamylase derived from Trametes cingulata shown in SEQ ID NO: 12 set forth herein, or in SEQ ID NO: 2 in WO 2006/69289.

[00221] AAPE096: Alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), shown in SEQ ID NO: 13 presented in this document, with the following substitutions: G128D+D143N.

[00222] Cellulase VD: Cellulatic composition derived from Trichoderma reesei further comprising GH61A polypeptide from Penicillium emersonii, disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 presented herein, and beta-glucosidase from Aspergillus fumigatus disclosed as SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 8 set forth herein, or a variant thereof, preferably a variant having one, preferably all, of the following substitutions: F100D, S283G, N456E, F512Y and Cel7A CBHI from Aspergillus fumigatus disclosed as SEQ ID NO: 6 in WO 2011/057140 and SEQ ID NO: 6 disclosed herein and CBH II from Aspergillus fumigatus disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 disclosed in this document.

[00223] Oxa Protease: Metalloprotease derived from Thermoascus aurantiacus disclosed as the mature part of SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature polypeptide of SEQ ID NO: 3 presented herein.

[00224] Mg protease 3: Protease from the S53 family of serine peptidases from a strain of Meripilus giganteus referred to in Example 2 in WO 2014/037438 or in SEQ ID NO: 20.

Methods: Identity

[00225] The relationship between two amino acid sequences or between two polynucleotide sequences is described by the "identity" parameter.

[00226] For purposes of the present invention, the degree of identity between two amino acid sequences is determined by the Clustal method (Higgins, 1989, CABIOS 5: 151-375+ wucpfq q uqhVyctg NAUGTIGPE™ OGIANKIP™ *FPCUTAR, Inc., Madison , WI) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and Gap length penalty of 10. Pairwise alignment parameters are Ktuple=1, Gap penalty=3, Windows=5 , and diagonals=5.

[00227] For the purposes of the present invention, the degree of identity between two polynucleotide sequences is determined by the Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA 80: 726-952+" wucpfq " q" uqhvyctg" NAUGTIGPG™ OGIANKIP™" *FPAUVAT." Kpe0." Ocfkuqp." YK+" eqo" woc" vcdgnc" fg identity and the following multi-align parameters: Gap penalty of 10 and Gap length penalty of 10. Pairwise alignment parameters are Ktuple=3, gap penalty=3, windows=20 SIGMA Enzyme Assay for Trealase

[00228] One SIGMA unit will convert 1.0 micromoles of trehalose into 2.0 micromoles of glucose per minute at pH 5.7 at 37 °C (released glucose determined at pH 7.5).

glucoamylase activity

[00229] Glucoamylase activity can be measured in Glucoamylase Units (AGU).

Glucoamylase activity (AGU)

[00230] The New Glucoamylase Unit (AGU) is defined as the amount of enzyme that hydrolyzes 1 micromole of maltose per minute under standard conditions 37 °C, pH 4.3, substrate: maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes.

[00231] An autoanalyzing system can be used. Mutarotase is added to the glucose dehydrogenase reagent such that any alpha-D-glucose present is transformed into beta-D-glucose. Glucose dehydrogenase specifically reacts with beta-D-glucose in the reaction mentioned above, forming NADH which is determined using a photometer at 340 nm as a measure of the original glucose concentration.

[00232] A folder (EB-SM-0131.02/01) describing this analytical method in more detail is available upon request from Novozymes A/S, Denmark, whose folder is included herein by reference.

Alpha-amylase activity (KNU)

[00233] Alpha-amylase activity can be determined using potato starch as substrate. This method is based on the breakdown of enzyme-modified potato starch, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially a blackish-blue color is formed, but during starch breakdown the blue color becomes weaker and gradually turns into a reddish-brown, which is compared with a stained-glass pattern.

[00234] A New Kilo alpha-amylase Unit (KNU) is defined as the amount of enzyme that, under standard conditions (i.e., at 37 °C +/0.05; Ca2+ at 0.0003 M; and pH 5, 6) dextrinizes 5.260 g Merck Amylum starch dry substance soluble.

[00235] A folder EB-SM-0009.02/01 describing this analytical method in more detail is available upon request from Novozymes A/S, Denmark, whose folder is included herein by reference.

Alpha-Amylase Acid Activity

[00236] When used in accordance with the present invention, the activity of an acid alpha-amylase can be measured in AFAU (fungal acid alpha-amylase units) or FAU-F.

Acid alpha-amylase activity (AFAU)

[00237] Acid alpha-amylase activity can be measured in AFAU (Fungal Acid Alpha-Amylase Units), which are determined relative to an enzyme standard. 1 AFAU is defined as the amount of enzyme that degrades 5.260 mg of starch dry matter per hour under the standard conditions mentioned below.

[00238] Acid alpha-amylase, an endo-alpha-amylase (1,4-alpha-D-glucan-glucanhydrolase, EC 3.2.1.1) hydrolyzes alpha-1,4-glycosidic bonds in the inner regions of the starch molecule, to form dextrins and oligosaccharides with different chain lengths. The intensity of the color formed with iodine is directly proportional to the starch concentration. Amylase activity is determined using reverse colorimetry as a reduction in starch concentration under the specified analytical conditions.

[00239] A folder EB-SM-0259.02/01 describing this analytical method in more detail is available upon request from Novozymes A/S, Denmark, whose folder is included herein by reference.

Determination of FAU-F

[00240] FAU-F Fungal Alpha-Amylase Units (Fungamil) is measured against an enzyme standard of a stated strength.

[00241] A folder (EB-SM-0216.02) describing this standard method in more detail is available upon request from Novozymes A/S, Denmark, whose folder is included in this document by reference. Measurement of Cellulase Activity Using Filter Paper Assay (FPU Assay) 1. Source of the Method 1.1 The method is disclosed in a document entitled "Measurement of Cellulase Activities" by Adney, B. and Baker, J. 1996. Laboratory Analytical Procedure , LAP-006, National Renewable Energy Laboratory (NREL). This is based on the IUPAC method for measuring cellulase activity (Ghose, T.K., Measurement of Cellulse Activities, Pure & Appl. Chem. 59, pg. 257-268, 1987. 2. Procedure 2.1 The method is carried out such as described by Adney and Baker, 1996, supra, except for the use of 96-well plates to read absorbance values after color development, as described below. coiled filter (#1 Whatman; 1 X 6 cm; 50 mg) is added to the bottom of a test tube (13 x 100 mm). 2.2.2 To the tube is added 1.0 ml of NA citrate buffer a 0.05 M (pH 4.80) 2.2.3 The tubes containing the filter paper and buffer are incubated for 5 min. at 50fl C (± 0.1 fl C) in a circulating water bath. 2.2.4 After After the incubation, 0.5 ml of enzyme dilution in citrate buffer is added to the tube. Enzyme dilutions are designed to produce values slightly above and below the target value of 2.0 mg of glucose. 2.2.5 The contents of the tubes mixed gently by vortexing for 3 seconds. 2.2.6 After vortexing, the tubes are incubated for 60 min. at 50 fl C (± 0.1fl C) in a circulating water bath. 2.2.7 Immediately after the 60 min. of incubation, the tubes are removed from the water bath, and 3.0 ml of DNS reagent is added to each tube to stop the reaction. The tubes are vortexed for 3 seconds to mix. 2.3 Blank and Control 2.3.1 A reagent blank is prepared by adding 1.5 ml of citrate buffer to a test tube. 2.3.2 A control substrate is prepared by placing a rolled-up strip of filter paper in the bottom of a test tube and adding 1.5 ml of citrate buffer. 2.3.3 Enzyme controls are prepared for each enzyme dilution by mixing 1.0 ml citrate buffer with 0.5 ml appropriate enzyme dilution. 2.3.4 Reagent Blank, Substrate Control, and Enzyme Controls are tested in the same way as and made in conjunction with enzyme test tubes. 2.4 Glucose Standards 2.4.1 A 100 ml glucose stock solution (10.0 mg/ml) is prepared, and 5 ml aliquots are frozen. Prior to use, aliquots are thawed and vortexed to mix. 2.4.2 Stock solution dilutions are performed in citrate buffer as follows: G1 = 1.0 ml stock + 0.5 ml buffer = 6.7 mg/ml = 3.3 mg/0.5 ml G2 = 0.75 ml stock + 0.75 ml buffer = 5.0 mg/ml = 2.5 mg/0.5 ml G3 = 0.5 ml stock + 1.0 ml buffer = 3.3 mg/ml = 1 .7 mg/0.5 ml G4 = 0.2 ml stock + 0.8 ml buffer = 2.0 mg/ml = 1.0 mg/0.5 ml 2.4.3 Glucose standard tubes are prepared by adding 0.5 ml of each dilution to 1.0 ml of citrate buffer. 2.4.4 Glucose standard tubes are tested in the same way as enzyme test tubes and made together with them. 2.5 Color Development 2.5.1 After 60 min. of incubation and addition of DNS, the tubes are all boiled for 5 mins. in a water bath. 2.5.2 After boiling, they are immediately cooled in an ice/water bath. 2.5.3 When cold tubes are briefly vortexed, and the pulp is allowed to settle. Then, each tube is diluted by adding 50 microl of the tube to 200 microl of ddH20 in a 96-well plate. Each well is mixed, and absorbance is read at 540 nm. 2.6 Calculations (examples are given in the NREL document) 2.6.1 A standard glucose curve is prepared by plotting the glucose concentration (mg/0.5 ml) for the four standards (G1-G4) vs. A540. This is fitted using linear regression (Prism Software), and the equation for the line is used to determine the glucose produced by each of the enzyme test tubes. 2.6.2 A plot of produced glucose (mg/0.5 ml) vs. the total enzyme dilution, with the Y-axis (enzyme dilution being a logarithmic scale. 2.6.3 A line is drawn between the enzyme dilution that produces just above 2.0 mg of glucose and the dilution that produces just below that From this line, the enzyme dilution is determined that would produce exactly 2.0 mg of glucose 2.6.4 Filter Paper Units/ml (FPU/ml) are calculated as follows: FPU/ml = 0.37 / enzyme dilution producing 2.0 mg of glucose

Protease Assay Method - AU(RH)

[00242] Proteolytic activity can be determined using denatured hemoglobin as substrate. In the Anson Hemoglobin method for determining proteolytic activity, denatured hemoglobin is digested, and undigested hemoglobin is precipitated with trichloroacetic acid (TCA). The amount of product soluble in TCA is determined with phenol reagent, which gives a blue color with tyrosine and tryptophan.

[00243] An Anson Unit (AU-RH) is defined as the amount of enzyme that under standard conditions (i.e., 25 °C, pH 5.5 and reaction time of 10 min.) digests hemoglobin at an initial rate such that an amount of product soluble in TCA is released per minute that gives the same color with phenol reagent as a milliequivalent of tyrosine.

[00244] The AU(RH) method is described in EAL-SM-0350 and is available from Novozymes A/S Denmark upon request. Protease Assay Method (LAPU) 1 Leucine Amino Peptidase Unit (LAPU) is the amount of enzyme that breaks down 1 microM of substrate per minute under the following conditions: 26 mM L-leucine-p-nitroanilide as substrate, Tris buffer a 0.1 M (pH 8.0), 37 °C, reaction time 10 minutes. LAPU is described in EB-SM-0298.02/01 available from Novozymes A/S Denmark upon request. Determination of Maltogenic Amylase Activity (MANU)

[00245] One MANU (New Maltogenic Amylase Unit) can be defined as the amount of enzyme required to release a micromole of maltose per minute at a concentration of 10 mg of maltotriose substrate (Sigma M 8378) per ml of citrate buffer a 0.1 M, pH 5.0 at 37°C for 30 minutes. EXAMPLES Example 1 Production of Crude Starch Ethanol Using Alpha Amylase Gt AMG and AAPE096

[00246] Finely ground raw starch paste (250 microns) was generated for this experiment. Approximately 405 g of yellow dent corn flour (obtained from Southeast Iowa Renewable Energy, LA, USA "ground in-house") was added to 595 g of tap water and the dry solids (DS) level was determined to be 35 ,00%. The wort was prepared to 500 ppm urea and 3 mg/l penicillin using solutions of 200 g/l urea and 1 g/l penicillin, respectively, and adjusted to pH 4.5. Approximately 5 g of the prepared worts were pipetted into each of the pre-weighed 15 ml centrifuge tubes, which had holes drilled in the top. The tubes were weighed again in order to determine the mass of the wort added.

[00247] Ngxgfwtc TGF UVCR™ hqk tgkftcVcfc. eqo 7.7 i fg ngxgfwtc placed in 100 ml of 32°C tap water for 30 minutes. While the yeast was soaking, each wort sample was dosed with glucoamylase (AMG) and alpha-amylase (AA) enzymes in the amounts and ratios according to the tables below.

[00248] Each treatment contained 6 replicates. Enzyme dosage was calculated using the following equation:

[00249] Water was dosed for each sample so that the total added volume of enzyme, water, yeast and acid (added at the end of the experiment) was 275 μl/5 g of sample. Rehydrated yeast was dosed to 100 μl of yeast solution in each sample. Samples were placed in a 32°C water bath for 88 hours. Each sample was vortexed morning and evening each day to ensure good mixing. At 72 hours and 88 hours of fermentation, three samples of each treatment were sacrificed for HPLC analysis. The sacrificed samples were dosed with 50 µl of 40% v/v H2SO4, vortexed, and centrifuged for 10 min. at 1462xg. The supernatant was filtered through a 0.45 syringe filter and this was used directly for HPLC analysis. HPLC samples were analyzed on the following system.

TABELA: AMG de Gama Estreita: estudo da razão da dose da proteína AA

Exemplo 2 Produção de Etanol de Amido em Bruto Usando Alfa-Amilase Gt AMG e AAPE096 -Resposta de Dosagem Com e Sem Celulase VD Adicionada[00250] The method quantifies the analytes using calibration standards for dextrin (DP4+), maltotriose, maltose, glucose, fructose, acetic acid, lactic acid, glycerol and ethanol. A 4-point calibration including the origin is used. TABLE: Wide Range AMG: AA protein dose ratio study

TABLE: Narrow Range AMG: AA protein dose ratio study

Example 2 Production of Crude Starch Ethanol Using Alpha-Amylase Gt AMG and AAPE096 -Dose Response With and Without Added VD Cellulase

Exemplo 3 Produção de Etanol de Amido em Bruto Usando Alfa-Amilase Gt AMG e AAPE096 - Com Celulase VD e Protease Oxa[00251] In this example, the ratio of AMG and AA was kept constant at 93:7 ratio of protein, respectively, and the total dose was increased as shown in the Table. Cellulase was added to the indicated treatments. Doses are stated per gram of DS. Table: Alpha Amylase Gt AMG and AAPE096 Dosage Response - With and Without VD Cellulase Added

Example 3 Production of Crude Starch Ethanol Using Alpha Amylase Gt AMG and AAPE096 - With Cellulase VD and Oxa Protease

Exemplo 4 Produção de Etanol de Amido em Bruto Usando Alfa-Amilase TcAMG e AAPE096 - Com Celulase VD e Protease Oxa[00252] In this example, the ratio of AMG and AA was held constant at 94:6 ratio of protein, respectively, at 147.9 μg total protein dosage. Additional enzyme(s) in the indicated amounts were added in separate treatments and the results obtained according to the Table below.

Example 4 Production of Crude Starch Ethanol Using TcAMG Alpha-Amylase and AAPE096 - With VD Cellulase and Oxa Protease

Exemplo 5 Produção de Etanol de Amido em Bruto Usando Alfa-Amilase Ps AMG e AAPE096[00253] In this example, the ratio of AMG and AA was held constant at 77:23 ratio of protein, respectively, at 116 μg total protein dosage. Additional enzyme(s) in the indicated amounts were added in separate treatments and the results obtained according to the Table below.

Example 5 Production of Crude Starch Ethanol Using Alpha-Amylase Ps AMG and AAPE096

[00254] Finely ground raw starch paste (250 microns) was generated for this experiment. Approximately 405 g of yellow dent corn flour (obtained from Southeast Iowa Renewable Energy, LA, USA "ground in-house") was added to 595 g of tap water and the dry solids (DS) level was determined to be 34 .71%. The wort was prepared to 500 ppm urea and 3 mg/l penicillin using solutions of 200 g/l urea and 1 g/l penicillin, respectively, and adjusted to pH 4.5. Approximately 5 g of the prepared worts were pipetted into each of the pre-weighed 15 ml centrifuge tubes, which had holes drilled in the top. The tubes were weighed again in order to determine the mass of the wort added.

[00255] Ngxgfwtc TGF UVCR™ hqk tgkftcVcfc. eqo 7.7 i fg ngxgfwtc placed in 100 ml of 32°C tap water for 30 minutes. While the yeast was soaking, each wort sample was dosed with glucoamylase (AMG) and alpha-amylase (AA) enzymes in the amounts and ratios according to the tables below. Each treatment contained 6 replications. Enzyme dosage was calculated using the following equation:

[00256] Water was dosed for each sample so that the total added volume of enzyme, water, yeast and acid (added at the end of the experiment) was 275 μl/5 g of sample. Rehydrated yeast was dosed to 100 μl of yeast solution in each sample. Samples were placed in a 32°C water bath for 88 hours. Each sample was vortexed morning and evening each day to ensure good mixing.

[00257] At 72 hours and 88 hours of fermentation, three samples of each treatment were sacrificed for analysis by HPLC. The sacrificed samples were dosed with 50 µl of 40% v/v H2SO4, vortexed, and centrifuged for 10 min. at 1462xg. The supernatant was filtered through a 0.45 syringe filter and this was used directly for HPLC analysis. HPLC samples were analyzed on the following system.

TABELA: AMG de Gama Estreita: estudo da razão da dose da proteína AA (mosto % DS = 34,17%)

Exemplo 6 Produção de Etanol de Amido em Bruto Usando Alfa-Amilase Ps AMG e AAPE096 -Resposta de Dosagem Com e Sem Celulase VD Adicionada[00258] The method quantifies the analytes using calibration standards for dextrin (DP4+), maltotriose, maltose, glucose, fructose, acetic acid, lactic acid, glycerol and ethanol. A 4-point calibration including the origin is used. TABLE: Wide Range AMG: AA protein dose ratio study

TABLE: Narrow Range AMG: AA protein dose ratio study (must % DS = 34.17%)

Example 6 Production of Crude Starch Ethanol Using PsAMG Alpha-Amylase and AAPE096 -Dose Response With and Without Added VD Cellulase

[00259] In this example, the ratio of AMG and AA was kept constant at 90:10 ratio of protein, respectively, and the total dose was increased as shown in the Table below. VD Cellulase and Oxa Protease were added to the indicated treatments. Doses are stated per µg of enzyme protein per gram of DS.

Tabela: Ps AMG e AAPE096 com ou sem Celulase VD e Protease Oxa adicionadas (mosto % DS = 34,56%)

Exemplo 7 Produção de Etanol de Amido em Bruto Usando Alfa-Amilase Ps AMG e AAPE096 e Protease 3 de Mg[00260] Table: Ps AMG and PE 96 Dosage Response With and Without Added VD Cellulase (must % DS = 35.49%)

Table: Ps AMG and AAPE096 with or without added VD Cellulase and Oxa Protease (must % DS = 34.56%)

Example 7 Production of Crude Starch Ethanol Using Alpha Amylase Ps AMG and AAPE096 and Mg Protease 3

Tabela: Esquema de Dosagem[00261] Approximately 795 g of yellow dent corn (obtained from Lincolnway Ethanol; Turkish ground in-house milling grade in Bunn coffee grinder) was added to 1205 g of tap water and the dry solids (DS) level , was determined to be 34.64% (see below for method). This mixture was supplemented with 3 ppm of penicillin and 500 ppm of urea. The slurry was adjusted to pH 4.5 with 40% H 2 SO 4 . Approximately 5 g of this slurry was added to 15 ml tubes. Each tube was dosed with enzymes from ceqtfq eqo c Vcdgnc 3. ugiwkpfq rqt 322 μn fg ngxgfwtc tgkftcVcfc *7.7 i fg Fermentis Ethanol Red yeast in 100 ml H2O, incubated for 30 min at 32 °C with magnetic stirring). Table 2 shows the enzymes used for this experiment. Water was added to each tube to bring the total volume added (enzyme + water) to 5.3% of the initial wort weight. This volume correction causes all tubes in the experiment to have the same total percent solids, making ethanol concentrations directly comparable across treatments.

Table: Dosage Scheme

[00262] Actual enzyme dosages were based on the exact weight of corn paste in each tube according to the following formula:

[00263] The tubes were incubated at 32 °C and 6 repeated fermentations of each treatment were performed. All tubes were shaken at 24, 48 and 70 hours. Three samples were sacrificed for HPLC analysis, at 72 hours, and 3 at 88 hours. The HPLC preparation consisted of stopping the reaction by adding 50 µl of 40% H2SO4, centrifuging for 10 min at 1462xg, and filtering through a 0.45 µm filter. Samples were stored at 4°C. HPLC analysis HPLC system - Agilent's 1100/1200 series with Chem station software Column - Bio-Rad HPX-87H Ion Exclusion Column 300mm x 7.8mm parts# 125-0140 Bio-Rad guard cartridge cation H parts# 125- 0129, Support Parts# 125-0131 Method - 0.005 M H2SO4 Mobile Phase Flow Rate 0.6 ml/min Column Temperature - 65 °C IR Detector Temperature - 55 °C

[00264] The method quantifies the analytes using calibration standards for DP4+, DP3, DP2, glucose, fructose, acetic acid, lactic acid, glycerol and ethanol. A four-point calibration including the origin is used. Dry Solids Analysis

[00265] Approximately 3-5 g of corn paste sample is placed in a pre-weighed AI tray and the tray with the sample is placed in a Mettler Toledo HB43-S halogen moisture analyzer (Mettler-Toledo, Columbus, OH ). The hydration balance settings were as follows - Drying Program: Standard; Nominal temperature: 160 °C; power off mode: free; Time Interval: 30 sec.

[00266] Data were analyzed in JMP (SAS, Cary, NC). Treatments were compared to control with the Tukey-Kramer Honestly Significant Difference test (p < 0.05).

[00267] Table: Percent change in ethanol produced for each protease treatment compared to the control sample (0.5AGU/g DS TcAMG/AAPE096) after 72 and 88 hours of fermentation. Conclusions

[00268] Proteases help in increasing fermentation kinetics and overall final yields of ethanol. Mg Protease 3 significantly improves ethanol production and faster fermentation rates compared to TcAMG/PE096, PsAMG/AAPE096 (No Protease) and PsAMG/AAPE096 + Oxa Protease. The present invention is further described in the following numbered paragraphs: 1. An enzyme composition comprising glucoamylase and alpha-amylase, and optionally protease. 2. An enzyme composition of paragraph 1 comprising an i) glucoamylase; ii) alpha-amylase; iii) cellulotic enzyme composition; iv) optionally protease. 3. The enzyme composition of paragraph 2, wherein the cellulolytic enzyme composition is derived from a strain of Trichoderma, such as Trichoderma reesei; a Humicola strain, such as Humicola insolens; a strain of Chrysosporium, such as Chrysosporium lucknowense; or a strain of Penicillium, such as Penicillium decumbens. 4. The enzyme composition of paragraphs 2 or 3, wherein the cellulolytic enzyme composition is derived from a strain of richoderma reesei and Trichoderma reesei comprises a beta-glycosidase, a cellobiohydrolase, and an endoglucanase. 5. The enzyme composition of any one of paragraphs 1-4, wherein a cellulolytic enzyme composition comprises one or more polypeptides selected from the group consisting of: - beta-glucosidase; - cellobiohydrolase I; - cellobiohydrolase II; or a mixture thereof. 6. The enzyme composition of any one of paragraphs 1-5, wherein the cellulolytic enzyme composition further comprises a GH61 polypeptide. 7. The enzyme composition of any one of paragraphs 1-6, wherein the cellulolytic enzyme composition comprises a beta-glucosidase, preferably one derived from a strain of the genus Aspergillus, such as Aspergillus oryzae, such as that disclosed in WO 2002 /095014 or the fusion protein having beta-glucosidase activity disclosed in WO 2008/057637, or Aspergillus fumigatus, such as one disclosed in SEQ ID NO: 2 in WO 2005/047499 or in SEQ ID NO: 6 set forth herein or an Aspergillus fumigatus beta-glucosidase variant disclosed in WO 2012/044915; or a strain of the genus Penicillium, such as a strain of Penicillium brasilianum disclosed in WO 2007/019442, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei. 8. The enzyme composition of any one of paragraphs 1-7, wherein the beta-glucosidase is from an Aspergillus strain, such as an Aspergillus fumigatus strain, such as Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 8 disclosed herein), or a variant thereof, which variant comprises one or more substitutions selected from the group consisting of L89M, G91L, F100D, I140V, I186V, S283G, N456E, and F512Y; as a variant of the same with the following replacements: - F100D + S283G + N456E + F512Y; - L89M + G91L + I186V + I140V; - I186V + L89M + G91L + I140V + F100D + S283G + N456E + F512Y. 9. The enzyme composition of any one of paragraphs 1-8, wherein the parent beta-glucosidase has at least 60% identity, such as at least 70%, such as at least 80%, such as at least 90% , such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity with the mature polypeptide of SEQ ID NO: 8 set forth herein . 10. The enzyme composition of any one of paragraphs 19, wherein the parent beta-glucosidase variant has at least 60% identity, such as at least 70%, such as at least 80%, such as at least 90% , such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, but less than 100% identity with the mature polypeptide of SEQ ID NO: 8 presented in this document. 11. The enzyme composition of any one of paragraphs 119, wherein the cellulotic enzyme composition comprises a GH61 polypeptide, such as one derived from the genus Thermoascus, such as a strain of Thermoascus aurantiacus, as described in WO 2005/ 074656 as SEQ ID NO: 2 or SEQ ID NO: 9 in this document; or a derivative of the genus Thielavia, such as a strain of Thielavia terrestris, as described in WO 2005/074647 as SEQ ID NO: 7 and SEQ ID NO: 8; or a derivative of a strain of Aspergillus, such as a strain of Aspergillus fumigatus, as described in WO 2010/138754 as SEQ ID NO: 1 and SEQ ID NO: 2; or a derivative of a derived strain of Penicillium, such as a strain of Penicillium emersonii, as disclosed in WO 2011/041397 as SEQ ID NO: 10 set forth herein. 12. The enzyme composition of paragraph 11, wherein the GH61 polypeptide, such as GH61 polypeptide from Penicillium sp. is selected from the group consisting of: (i) a GH61 polypeptide comprising the mature polypeptide of SEQ ID NO: 10 set forth herein; (ii) a GH61 polypeptide comprising an amino acid sequence having at least 60%, such as at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 10 presented in this document. 13. The enzyme composition of any one of paragraphs 112, wherein the cellulolytic enzyme composition comprises a cellobiohydrolase I (CBH I), such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as the CBH I Cel7 disclosed herein in SEQ ID NO: 6 in WO 2011/057140 or in SEQ ID NO: 6 set forth herein, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei. 14. The enzyme composition of any one of paragraphs 113, wherein the cellobiohydrolase I, such as cellobiohydrolase I from Aspergillus fumigatus, is selected from the group consisting of: (i) a cellobiohydrolase I comprising the mature polypeptide of SEQ ID NO: 6 presented in this document; (ii) a cellobiohydrolase I comprising an amino acid sequence having at least 70%, e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 89%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98% or at least 99% identity with the mature polypeptide of SEQ ID NO: 6 presented herein. 15. The enzyme composition of any one of paragraphs 114, wherein the cellulolytic enzyme composition comprises a cellobiohydrolase II (CBH II), such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as the one disclosed herein as SEQ ID NO: 7 shown herein; or a strain of the genus Trichoderma, such as Trichoderma reesei, or a strain of the genus Thielavia, such as a strain of Thielavia terrestris, such as cellobiohydrolase II CEL6A of Thielavia terrestris. 16. The enzyme composition of paragraphs 15, wherein the cellobiohydrolase II, such as cellobiohydrolase II from Aspergillus fumigatus, is selected from the group consisting of: (i) a cellobiohydrolase II comprising the mature polypeptide of SEQ ID NO: 7 presented in this document; (ii) a cellobiohydrolase II comprising an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 89%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98% or at least 99% identity with the mature polypeptide of SEQ ID NO: 7 set forth herein. 17. The enzyme composition of any one of paragraphs 116, wherein the cellulolytic enzyme composition comprises a GH61 polypeptide having increased cellulolytic activity and a beta-glucosidase. 18. The enzyme composition of any one of paragraphs 117, wherein the cellulolytic enzyme composition comprises a GH61 polypeptide having increased cellulolytic activity and a beta-glucosidase. 19. The enzyme composition of any one of paragraphs 118, wherein the cellulolytic enzyme composition comprises a GH61 polypeptide having increased cellulolytic activity, a beta-glucosidase and a CBHI. 20. The enzyme composition of any one of paragraphs 119, wherein the cellulolytic enzyme composition comprises a GH61 polypeptide having increased cellulolytic activity, a beta-glucosidase, a CBHI, and a CBHII. 21. The enzyme composition according to any one of paragraphs 1-20, wherein the cellulolytic enzyme composition is a cellulolytic composition from Trichoderma reesei, further comprising a GH61A polypeptide from Thermoascus aurantiacus (SEQ ID NO: 2 in WO 2005/ 074656 or SEQ ID NO: 9 shown herein), and Aspergillus oryzae beta-glucosidase fusion protein (WO 2008/057637). 22. The enzyme composition according to any one of paragraphs 1-21, wherein the cellulolytic enzyme composition is a cellulolytic composition from Trichoderma reesei, further comprising a GH61A polypeptide from Thermoascus aurantiacus having increased cellulolytic activity (SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 9 presented herein) and Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499) or SEQ ID NO: 8 disclosed herein. 23. The enzyme composition of any one of paragraphs 122, wherein the cellulolytic enzyme composition is a Trichoderma reesei cellulolytic composition further comprising Penicillium emersonii GH61A polypeptide disclosed in WO 2011/041397 or SEQ ID NO: 10 disclosed herein ) and beta-glucosidase from Aspergillus fumigatus (SEQ ID NO: 2 of WO 2005/047499) or SEQ ID NO: 8 presented herein) or a variant thereof, which variant has the following substitutions: F100D, S283G, N456E, F512Y , and optionally Aspergillus fumigatus CBHI disclosed as SEQ ID NO: 6 in WO 2011/057140 and SEQ ID NO: 6 disclosed herein and Aspergillus fumigatus CBH II disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 presented in this document. 24. The enzyme composition of any one of paragraphs 14, wherein a cellulolytic enzyme composition comprises one or more polypeptides selected from the group consisting of: (i) a cellobiohydrolase I from Aspergillus fumigatus; (ii) a cellobiohydrolase II from Aspergillus fumigatus; (iii) an Aspergillus fumigatus beta-glucosidase or a variant thereof. 25. The enzyme composition of paragraph 24, wherein beta glucosidase from Aspergillus fumigatus (SEQ ID NO: 8 shown herein), comprising one or more substitutions selected from the group consisting of f L89M, G91L, F100D, I140V, I186V , S283G, N456E, and F512Y; as a variant thereof with the following replacements: - F100D + S283G + N456E + F512Y; - L89M + G91L + I186V + I140V; - I186V + L89M + G91L + I140V + F100D + S283G + N456E + F512Y. 26. The cellulolytic composition of paragraphs 24 or 25 further comprises the GH61 polypeptide from Penicillium sp. shown in SEQ ID NO: 10 set forth in this document; or a GH61 polypeptide comprising an amino acid sequence having at least 60%, such as at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91% , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO : 10 presented in this document. 27. The enzyme composition of any one of paragraphs 125, wherein the glucoamylase is derived from a strain of Trametes, such as Trametes cingulata; or Pachykytospora, such as Pachykytospora papyracea; or Leucopaxillus, such as Leucopaxillus giganteus. 28. The enzyme composition of any one of paragraphs 127, wherein the glucoamylase, such as glucoamylase from Trametes cingulata, is selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 12 presented in this document; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 12 presented in this document. 29. The enzyme composition of any one of paragraphs 128, wherein the glucoamylase is from a strain of Aspergillus, preferably A. niger, A. awamori, or A. oryzae; or a strain of Trichoderma, preferably Trichoderma reesei; or a strain of Talaromyces, preferably Talaromyces emersonii. 30. The enzyme composition of any one of paragraphs 129, wherein the glucoamylase, such as Talaromyces emersonii glucoamylase, is selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 11 presented in this document; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 11 presented in this document. 31. The enzyme composition of paragraph 32, wherein the glucoamylase is derived from a strain of Penicillium, such as a strain of Penicillium oxalicum. 32. The enzyme composition of any one of paragraphs 132, wherein the glucoamylase, such as glucoamylase from Penicillium oxalicum, is selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 16 presented in this document; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 16 presented in this document. 33. The enzyme composition of any one of paragraphs 132, wherein the glucoamylase is derived from a strain of Gloeophyllum, such as a strain of Gloeophyllum sepiarium, such as that shown in SEQ ID NO: 2 in WO 2011/068803 or in SEQ ID NO: 4 shown in this document. 34. The enzyme composition of any one of paragraphs 133, wherein the glucoamylase, such as glucoamylase from Gloeophyllum sepiarium, is selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 4 presented in this document; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 2 in WO 2011/068803 or SEQ ID NO: 4 set forth herein. 35. The enzyme composition of any one of paragraphs 134, wherein the glucoamylase is derived from a strain of Gloeophyllum, such as one derived from a strain of Gloeophyllum trabeum, such as that shown in SEQ ID NO: 18. 36. The enzyme composition of any one of paragraphs 1-35, wherein the glucoamylase is selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 18 set forth herein; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 18 presented in this document. 37. The enzyme composition of paragraph 36, wherein the glucoamylase is the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18, disclosed herein preferably having one or more of the following substitutions: S95P, A121P, especially S95P+ A21P. 38. The enzyme composition of paragraphs 1-37, comprising Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18, disclosed herein preferably having one or more of the following substitutions: S95P, A121P, especially S95P+ A21P, and a alpha-amylase derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably that disclosed as V039 in Table 5 in WO 2006/069290, or in SEQ ID NO: 13, shown herein, preferably one having one or more of the following substitutions: G128D, D143N, especially G128D+D143N. 39. The enzyme composition of paragraphs 1-38, wherein the glucoamylase is derived from Pycnoporus, preferably Pycnoporus sanguineus, as shown in SEQ ID NO: 17 set forth herein. 40. The enzyme composition of paragraphs 1-39, wherein the enzyme composition comprises Pycnoporus sanguineus glucoamylase shown in SEQ ID NO: 17 shown herein, and an alpha-amylase derived from a Rhizomucor pusillus with a glucoamylase linker of Aspergillus niger and starch binding domain (SBD), preferably the one disclosed as V039 in Table 5 in WO 2006/069290, or in SEQ ID NO: 13, presented in this document, preferably the one having one or more of the following substitutions: G128D, D143N, especially G128D+D143N. 41. The enzyme composition of any one of paragraphs 140, wherein the glucoamylase is selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 17 set forth herein; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 17 presented in this document. 42. The enzyme composition of any one of paragraphs 141, further comprising a pullulanase, such as one derived from Bacillus sp, such as that shown in SEQ ID NO: 15 set forth herein. 43. The enzyme composition of paragraph 42, wherein the pullulanase, such as pullulanase from Bacillus sp. is selected from the group consisting of: (i) a pullulanase comprising the mature polypeptide of SEQ ID NO: 15 set forth herein; (ii) a pullulanase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 15 presented in this document. 44. The enzyme composition of paragraphs 42 or 43, comprising a pullulanase, such as pullulanase from Bacillus deramificans, a glucoamylase from Talaromyces emersonii and/or glucoamylase from Gloeophyllum sepiarium. 45. The enzyme composition of any one of paragraphs 144, wherein the alpha-amylase is of fungal or bacterial origin. 46. The enzyme composition of any one of paragraphs 1-45, wherein the alpha-amylase is derived from a strain of the genus Rhizomucor, preferably a strain of Rhizomucor pusillus, such as that shown in SEQ ID NO: 3 in WO 2013/ 006756, or the genus Meripilus, preferably a strain of Meripilus giganteus. 47. The enzyme composition of any one of paragraphs 1-46, wherein the alpha-amylase is derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), as shown in SEQ ID NO: 13 shown herein, or an alpha-amylase selected from the group consisting of: (i) an alpha-amylase comprising the mature polypeptide of SEQ ID NO: 13 shown herein; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 13 presented in this document. 48. The enzyme composition of any one of paragraphs 45-48, wherein the alpha-amylase is a variant of the alpha-amylase shown in SEQ ID NO: 13 having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H + Y141W; G20S + Y141W; A76G + Y141W; G128D + Y141W; G128D + D143N; P219C + Y141W; N142D + D143N; Y141W + K192R; Y141W + D143N; Y141W + N383R; Y141W + P219C + A265C; Y141W + N142D + D143N; Y141W + K192R V410A; G128D + Y141W + D143N; Y141W + D143N + P219C; Y141W + D143N + K192R; G128D + D143N + K192R; Y141W + D143N + K192R + P219C; G128D + Y141W + D143N + K192R; or G128D + Y141W + D143N + K192R + P219C (using SEQ ID NO: 13 for numbering). 49. The enzyme composition of any one of paragraphs 148, wherein the alpha-amylase is derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably disclosed as SEQ ID NO: 13, shown herein preferably having one or more of the following substitutions: G128D, D143N, preferably G128D+D143N (using SEQ ID NO: 13 for numbering). 50. The enzyme composition of any one of paragraphs 149, wherein the protease is of fungal origin. 51. The enzyme composition of any one of paragraphs 150, wherein the protease is a metalloprotease derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670, such as the metalloprotease disclosed as the mature part of SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature polypeptide of SEQ ID NO: 3 disclosed herein. 52. The enzyme composition of any one of paragraphs 151, wherein the protease, such as Thermoascus aurantiacus protease, is selected from the group consisting of: (i) a protease comprising the mature polypeptide of SEQ ID NO: 3 presented in this document; (ii) a protease comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 3 presented in this document. 53. The enzyme composition of any one of paragraphs 152, wherein the protease is of bacterial origin. 54. The enzyme composition of any one of paragraphs 153, wherein the protease is derived from a strain of Pyrococcus, such as a strain of Pyrococcus furiosus, such as the protease shown in SEQ ID NO: 1 in US 6,358,726 or in SEQ ID NO: 5 presented in this document. 55. The enzyme composition of any one of paragraphs 154, wherein the protease, such as Pyrococcus furiosus protease, is selected from the group consisting of: (i) a protease comprising the mature polypeptide of SEQ ID NO: 5 presented in this document; (ii) a protease comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 5 presented in this document. 56. The enzyme composition of any one of paragraphs 155, wherein the protease is derived from Meripilus giganteus, such as protease 3 from Meripilus giganteus, shown in SEQ ID NO: 19 herein and SEQ ID No: 5 in WO 2014 /037438. 57. The enzyme composition of any one of paragraphs 156, wherein the protease, such as Meripilus giganteus protease 3, is selected from the group consisting of: (i) a protease comprising the mature polypeptide of SEQ ID NO: 19 presented in this document; (ii) a protease comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 19 presented in this document. 58. The enzyme composition of any one of paragraphs 157, wherein the protease is derived from Meripilus giganteus, such as protease 3 from Meripilus giganteus, (protease of the S53 family of peptidases) discussed in Example 2 in WO 2014/037438 and shown as SEQ ID NO: 20 shown in this document. 59. The enzyme composition of any one of paragraphs 158, wherein the protease, such as Meripilus giganteus protease 3, is selected from the group consisting of: (i) a protease comprising the mature polypeptide of SEQ ID NO: 20 presented in this document; (ii) a protease comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 20 presented in this document. 60. The enzyme composition of any one of paragraphs 159, wherein the composition comprises i) fungal glucoamylase; ii) fungal alpha-amylase; iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide, CBH I and CBH II beta-glucosidase; iv) optionally protease. 61. The enzyme composition of any one of paragraphs 160, wherein the composition comprises i) Trametes cingulata glucoamylase; ii) Rhizomucor pusillus alpha-amylase, or a variant thereof; iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide from Penicillium emersonii, beta-glucosidase from Aspergillus fumigatus with the following substitutions: F100D, S283G, N456E, F512Y, and optionally CBH I from Aspergillus fumigatus and CBH II from Aspergillus fumigatus; optionally iv) Thermoascus aurantiacus protease, or a variant thereof. 62. The enzyme composition of any one of paragraphs 161, wherein the composition comprises i) Trametes cingulata glucoamylase; ii) alpha-amylase from Rhizomucor pusillus, or a variant thereof, iii) cellulolytic enzyme composition derived from a cellulolytic composition strain Trichoderma reesei, further comprising a GH61 polypeptide from Penicillium emersonii, beta-glucosidase from Aspergillus fumigatus with the following substitutions : F100D, S283G, N456E, F512Y, and optionally Aspergillus fumigatus CBH I and Aspergillus fumigatus CBH II; optionally iv) Pyropoccus furiosus protease. 63. The enzyme composition of any one of paragraphs 1-62, further comprising an enzyme selected from the group of trehalase and pectinase, such as pectin lyase or polygalacturonase. 64. The enzyme composition of paragraph 63, wherein the trehalase is of fungal origin, such as derived from a strain of Trichoderma, such as Trichoderma reesei, as shown in SEQ ID NO: 14 set forth herein. 65. The enzyme composition of paragraphs 63 and 64, wherein the trehalase, such as Trichoderma reesei trehalase, is selected from the group consisting of: (i) a trehalase comprising the mature polypeptide of SEQ ID NO: 14 shown herein document; (ii) a trehalase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 14 presented in this document. 66. A process for producing a fermentation product from a starch-containing material, comprising: (i) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and (ii) fermentation using a fermentation organism; wherein the saccharification and/or fermentation is carried out in the presence of the following enzymes: glucoamylase and alpha-amylase, and optionally protease. 67. The process of paragraph 66, wherein the glucoamylase is Gloeophyllum glucoamylase, preferably Gloeophyllum trabeum glucoamylase. 68. The process of either paragraph 66 or 67, wherein the glucoamylase is the glucoamylase from Gloeophyllum trabeum shown in SEQ ID NO: 18 or a glucoamylase having at least 60%, at least 70%, e.g., at least at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 18 shown herein. 69. The process of any one of paragraphs 66-68, wherein the glucoamylase is Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 having one of the following substitutions: V59A; S95P; A121P; T119W; S95P+A121P; V59A+S95P; S95P+T119W; V59A+S95P+A121P; or S95P+T119W+A121P, especially S95P+A121P. 70. The process of any one of paragraphs 66-68, wherein the alpha-amylase is derived from Rhizomucor pusillus preferably with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably that disclosed as V039 in Table 5 in WO 2006/069290, or in SEQ ID NO: 13 shown therein. 71. The enzyme composition of any one of paragraphs 66-70, wherein the alpha-amylase is derived from Rhizomucor pusillus. 72. The process of any one of paragraphs 66-71, wherein the glucoamylase is selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 18 set forth herein; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 18 presented in this document. 73. The process of any one of paragraphs 66-72, wherein the alpha-amylase is Rhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably one having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S + Y141W; A76G + Y141W; G128D + Y141W; G128D + D143N; P219C + Y141W; N142D + D143N; Y141W + K192R; Y141W + D143N; Y141W + N383R; Y141W + P219C + A265C; Y141W + N142D + D143N; Y141W + K192R V410A; G128D + Y141W + D143N; Y141W + D143N + P219C; Y141W + D143N + K192R; G128D + D143N + K192R; Y141W + D143N + K192R + P219C; G128D + Y141W + D143N + K192R; or G128D + Y141W + D143N + K192R + P219C, especially G128D+D143N (using SEQ ID NO: 13 for numbering). 74. The process of any one of paragraphs 66-73, wherein the glucoamylase is the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18, shown herein having one of the following substitutions: S95P+A121P and the alpha-amylase is alpha -amylase from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably one having at least one of the following substitutions G128D+D143N (using SEQ ID NO: 13 for numbering). 75. The process of any one of paragraphs 66-74, wherein the glucoamylase is the Pycnoporus sanguineus glucoamylase shown in SEQ ID NO: 17 shown herein. 76. The process of any one of paragraphs 66-75, wherein the glucoamylase is selected from the group consisting of: (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 17 set forth herein; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 17 presented in this document. 77. The process of any one of paragraphs 66-76, wherein the glucoamylase is the glucoamylase from Pycnoporus sanguineus shown in SEQ ID NO: 17 shown herein, or a glucoamylase having at least 60%, at least 70%, e.g. e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17 shown herein, and the alpha-amylase is Rhizomucor pusillus with a glucoamylase linker from Aspergillus niger and starch binding domain (SBD), preferably the one disclosed as V039 in Table 5 in WO 2006/069290, or in SEQ ID NO: 13 shown in this document, or an alpha-amylase having at least 60%, at least 70 %, eg at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95 %, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13 shown herein, preferably one having one or more of the following substitutions: G128D, D143N, especially G128D+D143N. 78. The process of any one of paragraphs 66-77, wherein the ratio of glucoamylase to alpha-amylase is between 99:1 and 1:2, such as between 98:2 and 1:1, such as between 97 :3 and 2:1, such as between 96:4 and 3:1, such as 97:3, 96:4, 95:5, 94:6, 93:7, 90:10, 85:15, 83: 17 or 65:35 (mg PE glucoamylase: mg PE alpha amylase). 79. The process of any one of paragraphs 66-78, where the total dose of glucoamylase and added alpha-amylase is 10-1,000 μg/g DS, such as 50-500 μg/g DS, as such as 75-250 µg/g DS. 80. The process of any one of paragraphs 66-75, wherein the total dose of added cellulotic enzyme composition is 10-500 µg/g DS, such as 20-400 µg/g DS, such as 20 -300 μg/g DS. 81. The process of any one of paragraphs 66-77, wherein the added dose of protease is 1-200 µg/g DS, such as 2-100 µg/g DS, such as 3-50 µg/ g of DS. 82. The process of any one of paragraphs 66-77, wherein the protease is derived from Meripilus giganteus, such as protease 3 from Meripilus giganteus, shown in SEQ ID NO: 19 herein and SEQ ID NO: 5 in WO 2014/037438. 83. The process of any one of paragraphs 66-78, wherein the protease, such as Meripilus giganteus protease 3, is selected from the group consisting of: (i) a protease comprising the mature polypeptide of SEQ ID NO: 19 presented in this document; (ii) a protease comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 19 presented in this document. 84. The process of any one of paragraphs 66-79, wherein the protease is derived from Meripilus giganteus, such as protease 3 from Meripilus giganteus, (protease of the S53 family of peptidases) discussed in Example 2 in WO 2014/037438 and shown as SEQ ID NO: 20 shown in this document. 85. The process of any one of paragraphs 66-80, wherein the protease, such as Meripilus giganteus protease 3, is selected from the group consisting of: (i) a protease comprising the mature polypeptide of SEQ ID NO: 20 presented in this document; (ii) a protease comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the mature polypeptide of SEQ ID NO: 20 presented in this document. 86. A process for making any one of paragraphs 66-81, comprising: i) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and ii) fermentation using a fermentation organism; wherein the saccharification and/or fermentation is carried out in the presence of the following enzymes: iii) glucoamylase; iv) alpha-amylase; v) i) cellulotic enzyme composition; optionally iv) protease. 87. The process of any one of paragraphs 66-82, wherein an enzyme composition of any one of paragraphs 1-81 is added or present during saccharification and/or fermentation. 88. The process of any one of claims 66-83, wherein saccharification and fermentation are done separately or simultaneously (i.e., as a one-step process or no-bake process). 89. The process of any one of paragraphs 66-85, wherein the fermentation product, such as ethanol, is recovered after fermentation. 90. The process of any one of paragraphs 66-85, where the starch-containing material is plant material selected from corn, cobs, wheat, barley, rye, milo, sago, tapioca, cassava, sorghum, rice, peas , beans, sweet potatoes, or a mixture of these, preferably corn. 91. The process of any one of paragraphs 66-86, wherein the starch-containing material is granular starch. 92. The process of any one of paragraphs 66-86, wherein the process is carried out at a pH in the range between 3 and 7, preferably 3 to 6, or more preferably 3.5 to 5.0. 93. The process of any one of paragraphs 66-88, wherein the dry solids content (DS) is in the range 10-55% by weight (DS), preferably 25-45% by weight, more preferably 30- 40% starch-containing material. 94. The process of any one of paragraphs 66-89, wherein the sugar concentration is maintained at a level below about 6% by weight, preferably 3% by weight, during saccharification and fermentation, especially below 0 .25% by weight. 95. The process of any one of paragraphs 66-90, wherein a slurry comprising material containing particle-reduced starch and water is prepared prior to step (a). 96. The process of any one of paragraphs 66-91, wherein the starch-containing material is prepared by reducing the particle size of the starch-containing material, preferably by milling, such that at least 50% of the starch-containing material starch has a particle size of 0.1-0.5 mm. 97. The process of any one of paragraphs 66-92, in which starch-containing plant material is reduced in particle size, either by dry milling or wet milling, or using particle size emulsion technology. 98. The process of any one of paragraphs 66-93, wherein the fermentation is carried out for 30 to 150 hours, preferably 48 to 96 hours. 99. The process of any one of paragraphs 66-94, where the temperature during fermentation in step (b) or simultaneous saccharification and fermentation in steps (a) and (b) is between 25 °C and 40 °C preferably between 28°C and 36°C, such as between 28°C and 35°C, such as between 28°C and 34°C, such as about 32°C. 100. The process of any one of paragraphs 66-95, wherein additionally a protease is present during saccharification and/or fermentation. 101. The process of any one of paragraphs 66-96, wherein the glucoamylase is present and/or added in an amount of from 0.001 to 10 AGU/g DS, preferably from 0.01 to 5 AGU/g DS, especially 0.1 to 0.5 AGU/g DS. 102. The process of any one of paragraphs 66-97, wherein the glucoamylase is present and/or added in an amount of 0.001 to 1000 micrograms Enzyme protein/g DS. 103. The process of any one of paragraphs 66-98, wherein alpha-amylase is present and/or added in an amount of from 0.001 to 10 AFAU/g DS, preferably from 0.01 to 5 AFAU/g of DS, especially 0.3 to 2 AFAU/g DS or 0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS. 104. The process of any one of paragraphs 66-99, wherein alpha-amylase is present and/or added in an amount of 0.001 to 1000 micrograms of Enzyme protein/g of DS. 105. The process of any one of paragraphs 66-100, wherein the cellulolytic enzyme composition is present and/or added in an amount of 1-10,000 microgram PE/g DS, such as 2-5,000, such as 3 and 1000, such as 4 and 500 microgram PE/g DS. 106. The process of any one of paragraphs 66-101, wherein the cellulolytic enzyme composition is present and/or added in an amount in the range of 0.1-100 FPU per gram total solids (TS), preferably 0.5-50 FPU per gram of TS, especially 1-20 FPU per gram of TS. 107. The process of any one of paragraphs 66-102, wherein the protease is present and/or is added in an amount in the range of 1-1000 µg PE/g DS, such as 2-500 µg PE /g DS, such as 3-250 µg PE/g DS. 108. The process of any one of paragraphs 66-104, wherein the fermenting organism is yeast, such as yeast derived from a strain of the genus Saccharomyces, preferably a strain of Saccharomyces cerevisiae. 109. The process of any one of paragraphs 66-104, wherein the fermentation organism is added to the fermentation, such as yeast, so that the count per ml of fermentation medium is in the range of 105 to 1012, preferably from 107 to 1010, especially around 5x107. 110. The process of any one of paragraphs 66-105, comprising: (a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and (b) fermentation using a fermentation organism; wherein the saccharification and/or fermentation is carried out in the presence of the following enzymes: (i) glucoamylase derived from Trametes cingulata; (j) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), or a variant thereof. 111. The process of any one of paragraphs 66-106, comprising: i) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and ii) fermentation using a fermentation organism; wherein the saccharification and/or fermentation is carried out in the presence of the following enzymes: iii) glucoamylase derived from Trametes cingulata; iv) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), or a variant thereof; v) i) cellulolytic enzyme composition derived from a strain of Trichoderma reesei. optionally iv) a protease from Thermoascus aurantiacus, or a variant thereof and/or Pyrococcus furiosus, or Meripilus giganteus. 112. The process of any one of paragraphs 66-107, comprising: (a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and (b) fermentation using a fermentation organism; wherein saccharification and/or fermentation is carried out in the presence of the following enzymes: i) glucoamylase derived from Gloeophyllum trabeum shown in SEQ ID NO: 18 shown herein, or a glucoamylase having at least 60%, at least 70%, p .eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 18 shown herein having, preferably, at least one of the following substitutions: V59A; S95P; A121P; T119W; S95P+A121P; V59A+S95P; S95P+T119W; V59A+S95P+A121P; or S95P+T119W+A121P, especially S95P+A121P (using SEQ ID NO: 18 for numbering). ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), or an alpha-amylase having at least 60%, at least 70%, for example at least 75% , at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98%, or at least 99% identity with SEQ ID NO: 13 shown herein, preferably one having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S + Y141W; A76G + Y141W; G128D + Y141W; G128D + D143N; P219C + Y141W; N142D + D143N; Y141W + K192R; Y141W + D143N; Y141W + N383R; Y141W + P219C + A265C; Y141W + N142D + D143N; Y141W + K192R V410A; G128D + Y141W + D143N; Y141W + D143N + P219C; Y141W + D143N + K192R; G128D + D143N + K192R; Y141W + D143N + K192R + P219C; G128D + Y141W + D143N + K192R; or G128D + Y141W + D143N + K192R + P219C, especially G128D+D143N (using SEQ ID NO: 13 for numbering). 113. The process of any one of paragraphs 66-108, comprising: i) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and ii) fermentation using a fermentation organism; wherein saccharification and/or fermentation is carried out in the presence of the following enzymes: iii) glucoamylase derived from Gloeophyllum trabeum shown in SEQ ID NO: 18 or a glucoamylase having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18 preferably having at least one of the following substitutions: V59A; S95P; A121P; T119W; S95P+A121P; V59A+S95P; S95P+T119W; V59A+S95P+A121P; or S95P+T119W+A121P, especially S95P+A121P (using SEQ ID NO: 18 for numbering). iv) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), preferably one having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S + Y141W; A76G + Y141W; G128D + Y141W; G128D + D143N; P219C + Y141W; N142D + D143N; Y141W + K192R; Y141W + D143N; Y141W + N383R; Y141W + P219C + A265C; Y141W + N142D + D143N; Y141W + K192R V410A; G128D + Y141W + D143N; Y141W + D143N + P219C; Y141W + D143N + K192R; G128D + D143N + K192R; Y141W + D143N + K192R + P219C; G128D + Y141W + D143N + K192R; or G128D + Y141W + D143N + K192R + P219C, especially G128D+D143N (using SEQ ID NO: 13 for numbering). v) i) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, preferably a cellulolytic enzyme composition derived from Trichoderma reesei further comprising the GH61A polypeptide from Penicillium emersonii disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO : 10 disclosed herein, and Aspergillus fumigatus beta-glucosidase disclosed as SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 8 disclosed herein, or a variant thereof, preferably a variant having one, preferably all, of the following substitutions: F100D, S283G, N456E, F512Y and optionally CBHI Cel7A from Aspergillus fumigatus disclosed as SEQ ID NO: 6 in WO 2011/057140 and SEQ ID NO: 6 set forth herein and CBH II from Aspergillus fumigatus disclosed as SEQ ID NO : 18 in WO 2011/057140 and as SEQ ID NO: 7 shown herein; optionally iv) a protease from Thermoascus aurantiacus, or a variant thereof and/or Pyrococcus furiosus, or Meripilus giganteus. 114. The process of any one of paragraphs 66-109, comprising: (i) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and (ii) fermentation using a fermentation organism; wherein the saccharification and/or fermentation is carried out in the presence of the following enzymes: i) glucoamylase derived from Pycnoporus sanguineus shown in SEQ ID NO: 17 shown herein, or a glucoamylase having at least 60%, at least 70%, p .eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17 set forth herein; ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), or an alpha-amylase having at least 60%, at least 70%, for example at least 75% , at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98%, or at least 99% identity with SEQ ID NO: 13 shown herein, preferably one having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S + Y141W; A76G + Y141W; G128D + Y141W; G128D + D143N; P219C + Y141W; N142D + D143N; Y141W + K192R; Y141W + D143N; Y141W + N383R; Y141W + P219C + A265C; Y141W + N142D + D143N; Y141W + K192R V410A; G128D + Y141W + D143N; Y141W + D143N + P219C; Y141W + D143N + K192R; G128D + D143N + K192R; Y141W + D143N + K192R + P219C; G128D + Y141W + D143N + K192R; or G128D + Y141W + D143N + K192R + P219C, especially G128D+D143N (using SEQ ID NO: 13 for numbering). iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, preferably a cellulolytic composition derived from Trichoderma reesei further comprising the GH61A polypeptide from Penicillium emersonii disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 disclosed herein document, and Aspergillus fumigatus beta-glucosidase disclosed as SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 8 disclosed herein; or a beta-glucosity having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8 shown in this document , or a variant thereof, preferably a variant having one, preferably all, of the following substitutions: F100D, S283G, N456E, F512Y (using SEQ ID NO: 8 for numbering) and optionally CBHI Cel7A from Aspergillus fumigatus disclosed as SEQ ID NO : 6 in WO 2011/057140 and SEQ ID NO: 6 set forth herein and CBH II from Aspergillus fumigatus disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 set forth herein. optionally iv) a protease from Thermoascus aurantiacus, or a variant thereof and/or Pyrococcus furiosus, or Meripilus giganteus. 111. The process of any one of paragraphs 66-110, comprising: i) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and ii) fermentation using a fermentation organism; wherein saccharification and/or fermentation is carried out in the presence of the following enzymes: iii) glucoamylase derived from Pycnoporus sanguineus shown in SEQ ID NO: 17 shown herein, or a glucoamylase having at least 60%, at least 70%, p .eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17 set forth herein; iv) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch binding domain (SBD), having the following substitutions G128D+D143N (using SEQ ID NO: 13 shown herein for numbering); v) i) optionally cellulolytic enzyme composition derived from a strain of Trichoderma reesei; vi) a protease 3 from Meripilus giganteus, preferably that shown in SEQ ID NO: 20 or a protease having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 20. 112. The process of any one of paragraphs 66-111, wherein the ratio of glucoamylase to alpha-amylase is between 99:1 and 1:2, such such as between 98:2 and 1:1, such as between 97:3 and 2:1, such as between 96:4 and 3:1, such as 97:3, 96:4, 95:5, 94:6, 93:7, 90:10, 85:15, 83:17 or 65:35 (mg PE glucoamylase: mg PE alpha amylase). 113. The process of paragraphs 66-112, where saccharification and fermentation are carried out simultaneously. 114. The process of any one of paragraphs 66-113, wherein an enzyme composition of paragraphs 1-65 is used as the enzyme/enzyme composition in saccharification or fermentation or simultaneous saccharification and fermentation.

[00269] The invention described and claimed in this document is not to be limited in scope by the specific embodiments disclosed in this document, as these embodiments are intended to illustrate various aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the aforementioned description. Such modifications are also intended to be within the scope of the appended claims. In case of conflict, this disclosure, including definitions, will control.

[00270] Various references are cited in this document, the disclosures of which are incorporated by reference in their entireties.

Claims (10)

1. Enzyme composition, characterized in that it comprises glucoamylase and alpha-amylase wherein the glucoamylase consists of the amino acid sequence of SEQ ID NO: 17 having from 0 to 10 conservative amino acid substitutions, wherein the alpha-amylase consists of amino acid sequence of SEQ ID NO: 13 having one or more of the following substitutions: G128D, D143N or G128D+D143N and from 0 to 10 conservative amino acid substitutions. 2. Enzyme composition according to claim 1, characterized in that it additionally comprises a cellulolytic enzyme composition, wherein the cellulolytic composition comprises a beta-glucosidase, a cellobiohydrolase and an endoglucanase. 3. Enzyme composition according to claim 2, characterized in that the cellulolytic enzyme composition is derived from a strain of Trichoderma; a strain of Humicola; a strain of Chrysosporium; or a strain of Penicillium. 4. Enzyme composition according to claim 1, characterized in that the enzyme composition comprises a protease. 5. Enzyme composition according to claim 1, characterized in that it further comprises an enzyme selected from the group of trehalase and pectinase. 6. A process for producing a fermentation product from starch-containing material, comprising: (i) saccharifying a material, wherein the starch-containing material comprises corn; and (ii) fermentation using a yeast fermentation organism to produce the fermentation product, wherein the fermentation product comprises ethanol; wherein the saccharification and fermentation are carried out simultaneously at a temperature between 25 and 40 degrees Celsius in the presence of the enzyme composition as defined in claim 1. 7. Process according to claim 6, characterized in that additionally the saccharification and/or fermentation is carried out in the presence of a cellulolytic enzyme composition derived from a strain of Trichoderma, Humicola, Chrysosporium, or Penicillium. 8. Process according to claim 6, characterized in that the cellulolytic enzyme composition comprises a beta-glucosidase, a cellohydrobiolase and an endoglucanase. 9. Process according to claim 6, characterized in that the ratio between glucoamylase and alpha-amylase is between 99:1 and 1:2 (mg of PE glucoamylase: mg of PE alpha-amylase). 10. Enzyme composition, according to claim 1, characterized in that the enzyme composition comprises a protease.